WO2023198015A1 - Molécule de liaison à l'antigène se liant spécifiquement à psma et cd28 et son utilisation pharmaceutique - Google Patents

Molécule de liaison à l'antigène se liant spécifiquement à psma et cd28 et son utilisation pharmaceutique Download PDF

Info

Publication number
WO2023198015A1
WO2023198015A1 PCT/CN2023/087463 CN2023087463W WO2023198015A1 WO 2023198015 A1 WO2023198015 A1 WO 2023198015A1 CN 2023087463 W CN2023087463 W CN 2023087463W WO 2023198015 A1 WO2023198015 A1 WO 2023198015A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
acid sequence
psma
strand
Prior art date
Application number
PCT/CN2023/087463
Other languages
English (en)
Chinese (zh)
Inventor
石金平
杨昊
黎婷婷
应华
陶维康
Original Assignee
江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Publication of WO2023198015A1 publication Critical patent/WO2023198015A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present disclosure belongs to the field of biotechnology, and more specifically, the present disclosure relates to PSMA/CD28 antigen-binding molecules and their applications.
  • PSMA belongs to Glutamate carboxypeptidase II (Glutamate carboxypeptidase II, GCPII) and consists of 750 amino acids.
  • the extracellular part of PSMA consists of three domains: protease-like domain, apical domain and C-terminus. All three domains are involved in substrate binding. Among them, the protease-like domain and the apical domain directly bind to the substrate, and the C-terminus allows PSMA to form a dimer to function.
  • the expression level of PSMA in tumor tissue is 100-1000 times higher than that in normal prostate. Especially in poorly differentiated, metastatic and hormone-refractory cancers, the expression of PSMA is significantly increased.
  • PSMA is also expressed in the endothelial cells of capillaries in the tumor periphery and intratumoral areas of some malignant tumors, but is not expressed in blood vessels of normal tissues.
  • PSMA is related to tumor angiogenesis (Silver D.A. (1997) Clinical Cancer Research 3: 81-85). Recently, PSMA has been shown to be expressed in endothelial cells of tumor-associated neovasculature in colon, breast, bladder, pancreatic, renal, and melanoma cancers (Chang, S.S. (2004) Curr Opin Investig Drugs 5: 611- 5). Therefore, PSMA can be used not only as a drug against prostate cancer, but may also be applicable to other types of tumors.
  • T cell activation relies on the first signal provided by TCR activated by APC cells or other cell surface MHC-peptide complexes.
  • costimulatory factors are required to provide a second signal to completely activate T cells.
  • CD28 is a member of the immunoglobulin superfamily. In humans, CD28 is mainly expressed on the surface of T cells, and is also expressed in small amounts on other cells such as bone marrow stromal cells, plasma cells, neutrophils, and eosinophils. CD28 molecules can further activate the downstream NFAT, NF- ⁇ B and AP-1 signaling pathways by binding to CD80/CD86 molecules expressed on the surface of activated APC cells, promoting the activation and proliferation of T cells.
  • providing bispecific antibodies targeting PSMA/CD28 is an effective means to treat tumors and other diseases.
  • the present disclosure provides an antibody that specifically binds CD28 and PSMA antigen-binding molecules and an antibody that specifically binds CD28.
  • the present disclosure provides an antigen-binding molecule comprising at least one specific binding
  • the antigen-binding module of CD28 and at least one antigen-binding module that specifically binds to PSMA includes the heavy chain variable region CD28-VH and the light chain variable region CD28-VL
  • the specificity The antigen-binding module that binds PSMA includes the heavy chain variable region PSMA-VH and the light chain variable region PSMA-VL.
  • the antigen-binding molecule as described above, wherein:
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 35
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL contain CD28 in SEQ ID NO: 36, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115 respectively -amino acid sequences of LCDR1, CD28-LCDR2 and CD28-LCDR3, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL contain CD28-LCDR1, CD28-LCDR2 and CD28 in SEQ ID NO: 34, 72, 73, 74, 75, 76, 77, 78, 79 or 80 respectively -The amino acid sequence of LCDR3, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 37, 129 or 132
  • CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in the CD28-VL respectively comprise the amino acids of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 38, 138, 139, 141, 142 or 143 sequence, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, 46, 47, 48 or 49
  • the amino acid sequence, and CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in the CD28-VL respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 32 or 52.
  • the antigen-binding molecule as described above, wherein:
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 35
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 36, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL contain the amino acids of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 80 respectively. amino acid sequence, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33, and the CD28-VL in the CD28-VL.
  • CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 34, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 37
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 38, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 32;
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, and the CD28-VL in the CD28-VL.
  • CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 52.
  • the CD28-HCDR1, CD28-HCDR2, CD28-HCDR3, CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 are defined according to Kabat, IMGT, Chothia, AbM or Contact numbering rules.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR1 comprising SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92 or 93 amino acid sequence of CD28-LCDR2 and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 24; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14 and CD28- comprising the amino acid sequence of SEQ ID NO: 15 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, comprising the amino acid sequence of SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25 or 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 116 and the amino acid sequence comprising SEQ ID NO: 27 CD28-HCDR3 of the sequence, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28- comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 LCDR2 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42 amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or 43, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or 43. ID NO: 12 amino acid sequence of CD28-LCDR3.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 30 amino acid sequence of CD28-LCDR3.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42.
  • the amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 12
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42.
  • the amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 12 Amino acid sequence of CD28-LCDR3.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23 and CD28- comprising the amino acid sequence of SEQ ID NO: 24 LCDR3; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15.
  • HCDR3 has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 62 and CD28- comprising the amino acid sequence of SEQ ID NO: 18 LCDR3; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 27.
  • HCDR3 has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29 and CD28- comprising the amino acid sequence of SEQ ID NO: 30 LCDR3; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28- comprising the amino acid sequence of SEQ ID NO: 9 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 12 LCDR3.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 22, such as SEQ ID NO: 23, 81, CD28-LCDR2 represented by 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 and CD28-LCDR3 represented by SEQ ID NO: 24; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 16, CD28-LCDR2 as shown in SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and CD28-LCDR3 shown in SEQ ID NO: 18; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25 or 117, CD28-HCDR2 as shown in SEQ ID NO: 26 or 116, and CD28-HCDR1 as shown in SEQ ID NO: 27 HCDR3, and the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 28, CD28-LCDR2 as set forth in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 as set forth in SEQ ID NO: CD28-LCDR3 shown in 30; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 7, CD28-HCDR2 as shown in SEQ ID NO: 8 and CD28-HCDR2 as shown in SEQ ID NO: 9, 39, 40, 41 or 42 CD28-HCDR3 as shown, and the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 10, CD28-LCDR2 as shown in SEQ ID NO: 11 or 43, and CD28-LCDR2 as shown in SEQ ID NO: 12 CD28-LCDR3.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25, CD28-HCDR2 as shown in SEQ ID NO: 26 and CD28-HCDR3 as shown in SEQ ID NO: 27, and the CD28- VL contains CD28-LCDR1 as shown in SEQ ID NO: 28, CD28-LCDR2 as shown in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR3 as shown in SEQ ID NO: 30 ;or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25, CD28-HCDR2 as shown in SEQ ID NO: 116 and CD28-HCDR3 as shown in SEQ ID NO: 27, and the CD28- VL contains CD28-LCDR1 as shown in SEQ ID NO: 28, CD28-LCDR2 as shown in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR3 as shown in SEQ ID NO: 30 ;or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 117, CD28-HCDR2 as shown in SEQ ID NO: 26 and CD28-HCDR3 as shown in SEQ ID NO: 27, and the CD28- VL contains CD28-LCDR1 as shown in SEQ ID NO: 28, CD28-LCDR2 as shown in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR3 as shown in SEQ ID NO: 30 ;or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 117, CD28-HCDR2 as shown in SEQ ID NO: 116 and CD28-HCDR3 as shown in SEQ ID NO: 27, and the CD28- VL includes CD28-LCDR1 as set forth in SEQ ID NO: 28, CD28-LCDR2 as set forth in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR3 as set forth in SEQ ID NO: 30 .
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 7, CD28-HCDR2 as shown in SEQ ID NO: 8 and CD28 as shown in SEQ ID NO: 9, 39, 40, 41 or 42 -HCDR3, and the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 10, CD28-LCDR2 as set forth in SEQ ID NO: 11, and CD28-LCDR3 as set forth in SEQ ID NO: 12; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 7, CD28-HCDR2 as shown in SEQ ID NO: 8 and CD28 as shown in SEQ ID NO: 9, 39, 40, 41 or 42 -HCDR3, and the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 10, CD28-LCDR2 as set forth in SEQ ID NO: 43, and CD28-LCDR3 as set forth in SEQ ID NO: 12.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and
  • the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 22, CD28-LCDR2 as set forth in SEQ ID NO: 23, and CD28-LCDR3 as set forth in SEQ ID NO: 24; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and
  • the CD28-VL is CD28-LCDR1 set forth in SEQ ID NO: 16, CD28-LCDR2 set forth in SEQ ID NO: 62, and CD28-LCDR3 set forth in SEQ ID NO: 18; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25, CD28-HCDR2 as shown in SEQ ID NO: 26 and CD28-HCDR3 as shown in SEQ ID NO: 27, and
  • the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 28, CD28-LCDR2 as set forth in SEQ ID NO: 29, and CD28-LCDR3 as set forth in SEQ ID NO: 30; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO:7, CD28-HCDR2 as shown in SEQ ID NO:8 and CD28-HCDR3 as shown in SEQ ID NO:9, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 10, CD28-LCDR2 as shown in SEQ ID NO: 43, and CD28-LCDR3 as shown in SEQ ID NO: 12.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and the CD28- VL contains CD28-LCDR1 as shown in SEQ ID NO:22, CD28-LCDR2 as shown in SEQ ID NO:23, and CD28-LCDR3 as shown in SEQ ID NO:24.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and the CD28- VL is CD28-LCDR1 as shown in SEQ ID NO: 16, CD28-LCDR2 as shown in SEQ ID NO: 62, and CD28-LCDR3 as shown in SEQ ID NO: 18.
  • the antigen-binding molecule as described in any one of the preceding items are defined according to the Kabat numbering rule.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 26A, 29L, 69L, 71V, 78A and 93S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 with the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 with the amino acid sequence of SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 and a CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and the FR region of the CD28-VL comprises one or more amino acid substitutions selected from the group consisting
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25 or 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 116 and the amino acid sequence comprising SEQ ID NO: 27
  • the sequence of CD28-HCDR3, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 71V and 78A
  • the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 having the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 having the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 and CD28-LCDR3 having the amino acid sequence of SEQ ID NO: 30, and said
  • the FR region of CD28-VL contains one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44V and 73L; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and SEQ ID NO: 9, 39, 40, 41 or a CD28-HCDR3 with an amino acid sequence of 42, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S; and the CD28- VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or 43, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 12
  • the CD28-LCDR3 of the column; and the FR region of the CD28-VL contains one or more amino acid substitutions selected from the group consisting of 43S and 73F.
  • the antigen-binding molecule as described in any one of the preceding items binds to human CD28 with a KD of less than 2 ⁇ 10 -8 M, 8 ⁇ 10 -9 M or 5 ⁇ 10 -9 M at 25°C. , the KD is measured by surface plasmon resonance method.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 26A, 29L, 69L, 71V, 78A and 93S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 23, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 24, and the FR region of CD28-VL includes the selected One or more amino acid substitutions from the group consisting of 41D, 42G, 43T, 44I and 71Y; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15.
  • the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 69L, 71V, 73K and 94S; and the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 62, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 62, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 18, and the FR region of the CD28-VL contains a protein selected from the group consisting of 43S and one or more amino acid substitutions in the group consisting of 70K; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 27.
  • CD28-VL has: the amino acid sequence comprising SEQ ID NO: 28 CD28-LCDR1, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30, and the FR region of the CD28-VL comprises 41D, 42G, 43T One or more amino acid substitutions in the group consisting of , 44V and 73L; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28- comprising the amino acid sequence of SEQ ID NO: 9 HCDR3, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S; and the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 having the amino acid sequence of 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 12; and said The FR region of CD28-VL contains one or more amino acid substitutions selected from the group consisting of 43S and 73F.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 164, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 165, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 166 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 167, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 168, and PSMA comprising the amino acid sequence of SEQ ID NO: 169 -LCDR3; or
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 170, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 171, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 172 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 173, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 174, and PSMA comprising the amino acid sequence of SEQ ID NO: 175 -LCDR3; or
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 158, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 159, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 161, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 162, and PSMA comprising the amino acid sequence of SEQ ID NO: 163 -LCDR3; or
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 176, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 177, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 178 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 179, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 180, and PSMA comprising the amino acid sequence of SEQ ID NO: 181 -LCDR3; or
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 213, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 214, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 215 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 216, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 217, and PSMA comprising the amino acid sequence of SEQ ID NO: 218 -LCDR3.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 170, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 171, and PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 172; And the PSMA-VL has: PSMA-LCDR1 including the amino acid sequence of SEQ ID NO: 173, PSMA-LCDR2 including the amino acid sequence of SEQ ID NO: 174, and PSMA-LCDR3 including the amino acid sequence of SEQ ID NO: 175 .
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH has: PSMA-HCDR1 including the amino acid sequence of SEQ ID NO: 164, PSMA-HCDR2 including the amino acid sequence of SEQ ID NO: 165, and PSMA-HCDR3 including the amino acid sequence of SEQ ID NO: 166; And the PSMA-VL has: PSMA-LCDR1 including the amino acid sequence of SEQ ID NO: 167, PSMA-LCDR2 including the amino acid sequence of SEQ ID NO: 168, and PSMA-LCDR3 including the amino acid sequence of SEQ ID NO: 169 .
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and Said PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 167, PSMA-LCDR2 as shown in SEQ ID NO: 168, and PSMA-LCDR3 as shown in SEQ ID NO: 169; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and PSMA-VL includes PSMA-LCDR1 as set forth in SEQ ID NO: 173, PSMA-LCDR2 as set forth in SEQ ID NO: 174, and PSMA-LCDR3 as set forth in SEQ ID NO: 175; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 158, PSMA-HCDR2 as shown in SEQ ID NO: 159, and PSMA-HCDR3 as shown in SEQ ID NO: 160; and PSMA-VL includes PSMA-LCDR1 as set forth in SEQ ID NO: 161, PSMA-LCDR2 as set forth in SEQ ID NO: 162, and PSMA-LCDR3 as set forth in SEQ ID NO: 163; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 176, PSMA-HCDR2 as shown in SEQ ID NO: 177, and PSMA-HCDR3 as shown in SEQ ID NO: 178; and Said PSMA-VL includes PSMA-LCDR1 as set forth in SEQ ID NO: 179, PSMA-LCDR2 as set forth in SEQ ID NO: 180, and PSMA-LCDR3 as set forth in SEQ ID NO: 181; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 213, PSMA-HCDR2 as shown in SEQ ID NO: 214, and PSMA-HCDR3 as shown in SEQ ID NO: 215; and
  • the PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 216, PSMA-LCDR2 as shown in SEQ ID NO: 217, and PSMA-LCDR3 as shown in SEQ ID NO: 218.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and the PSMA- VL contains PSMA-LCDR1 as shown in SEQ ID NO: 173, PSMA-LCDR2 as shown in SEQ ID NO: 174, and PSMA-LCDR3 as shown in SEQ ID NO: 175.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and
  • the PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO:167, PSMA-LCDR2 as shown in SEQ ID NO:168, and PSMA-LCDR3 as shown in SEQ ID NO:169.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 98 or a variant thereof, wherein the variant is in the FR region of SEQ ID NO: 98 and includes a sequence selected from 26A, 29L, 69L, 71V, 78A and 93S
  • One or more amino acid substitutions in the group consisting of and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 102 or a variant thereof, the variant being in the FR region of SEQ ID NO: 102 and comprising 41D selected from the group consisting of One or more amino acid substitutions from the group consisting of , 42G, 43T, 44I and 71Y; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 69 or a variant thereof, wherein the variant contains a sequence selected from the group consisting of 28S, 69L, 71V, 73K and 94S in the FR region of SEQ ID NO: 69 One or more amino acid substitutions in the group, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70 or a variant thereof, the variant being in the FR region of SEQ ID NO: 70 and including 43S and 70K One or more amino acid substitutions in the group consisting of; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 133 or a variant thereof, wherein the variant is one selected from the group consisting of 25P, 71V and 78A in the FR region of SEQ ID NO: 133 Or multiple amino acid substitutions, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 137 or a variant thereof, the variant is in the FR region of SEQ ID NO: 137.
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 53 or a variant thereof, wherein the variant contains a FR region selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S of SEQ ID NO: 53 consisting of one or more amino acid substitutions in the group, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52 or a variant thereof, the variant being in the FR region of SEQ ID NO: 52 and comprising 43S selected from the group consisting of One or more amino acid substitutions from the group consisting of and 73F.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 35, 94, 96, 97 or 98
  • the CD28-VL comprises SEQ ID NO: 101, 36, 99, 100, 102, 103 , 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115 amino acid sequence; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, 33, 63, 64, 65, 66, 67 or 69
  • the CD28-VL comprises SEQ ID NO: 80, 34, 70, 71 , 72, 73, 74, 75, 76, 77, 78 or 79 amino acid sequence
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, 37, 123, 124, 125, 127, 128, 129, 130, 131, 132 or 133
  • the CD28-VL comprises SEQ ID NO. : the amino acid sequence of 134, 38, 135, 136, 137, 138, 139, 140, 141, 142 or 143; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, 31, 45, 46, 47, 48, 49 or 53
  • the CD28-VL comprises SEQ ID NO: 52, 32, 50 or 51 amino acid sequence.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98
  • the CD28-VL comprises SEQ ID NO: 101, 99, 100, 102, 103, 104, 105 , the amino acid sequence of 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, 63, 64, 65, 66, 67 or 69
  • the CD28-VL comprises SEQ ID NO: 80, 70, 71, 72, 73 , 74, 75, 76, 77, 78 or 79 amino acid sequence
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, 123, 124, 125, 127, 128, 129, 130, 131, 132 or 133
  • the CD28-VL comprises SEQ ID NO: 134 , 135, 136, 137, 138, 139, 140, 141, 142 or 143 amino acid sequence
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, 45, 46, 47, 48, 49 or 53
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52, 50 or 51.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101, 99, 100 or 102, or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95
  • the CD28-VL includes SEQ ID NO: 101, 99, 100, 103, 104, 105, 106, 107, 108, 109, 110, 111, the amino acid sequence of 112, 113, 114 or 115, or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 94
  • the CD28-VL includes SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, the amino acid sequence of 112, 113, 114 or 115, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 96
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99 or 100, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 97
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99, 100 or 101, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 80, 72, 73, 74, 75, 76, 77, 78 or 79, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 63
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70 or 71, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 64
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71 or 72, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 65
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71 or 72, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 66
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71, 72, 74, 75, 76 or 79, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 67
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 74, 75, 76 or 79, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135, 136, 137, 138 or 139, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 123
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135 or 136, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 124, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 135; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 125
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134 or 135, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 127
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 137, 138 or 139; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 128, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 137, 138, 139 or 142; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 129
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 138, 139, 140, 141, 142 or 143, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 130
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 131
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52, 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 45
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 46
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 52, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 47
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 48
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 36;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 33
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 34;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 37
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 38;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 31, and the CD28-VL contains Contains the amino acid sequence of SEQ ID NO: 32.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 46
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 65
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 71
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 78; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 80; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 106; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 94
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134;
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 125, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 135; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 128, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 142; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 129
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 44, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 52; or
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 46, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 52; or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 68, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 78; or
  • CD28-VH is shown in SEQ ID NO: 68
  • amino acid sequence of CD28-VL is shown in SEQ ID NO: 80; or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 95, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 106; or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 94, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 101; or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 125, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 135; or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 128, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 142; or
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 129, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 138.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50, 51 or 52, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 45
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 46
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 52, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 47
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 48
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 63, 64, 65 or 66
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 63, 64, 65 or 66
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 71; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 64 or 65
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 66
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 71, 72, 74, 75, 76 or 79; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 67
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 74, 75, 76 or 79; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 73, 74, 75, 76, 78, 79 or 80; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 94
  • the CD28-VL comprises SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110 , 111, 112, 113, 114 or 115 amino acid sequence; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95
  • the CD28-VL includes SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, The amino acid sequence of 112, 113, 114 or 115; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 96
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99 or 100; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 97
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99, 100 or 101; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 123
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135 or 136;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 124, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 135; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 125
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134 or 135, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135, 136, 137, 138 or 139; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 127
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 137, 138 or 139; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 128, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 137, 138, 139 or 142; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 129
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 138, 139, 140, 141, 142 or 143; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 130
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 131
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, and the CD28-VL contains Containing the amino acid sequence of SEQ ID NO: 101; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 80
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the antigen-binding molecule as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 95, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 101 shown.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 68, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 80 shown.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, 199, 332, 194, 195, 196 or 198
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201, 202, 333 or 200 ;
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, 184 or 203
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206, 185 or 205; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188, 182, 189 or 190
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191, 183, 192 or 193; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 207 or 186
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 208, 187, 209 or 210; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 211
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212;
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201;
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 202; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191;
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 207
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 208.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH includes the amino acid sequence of SEQ ID NO:204, and the PSMA-VL includes the amino acid sequence of SEQ ID NO:206.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 202.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • amino acid sequence of the PSMA-VH is as shown in SEQ ID NO: 204
  • amino acid sequence of the PSMA-VL is as shown in SEQ ID NO: 206; or
  • amino acid sequence of the PSMA-VH is as shown in SEQ ID NO: 197
  • amino acid sequence of the PSMA-VL is as shown in SEQ ID NO: 201; or
  • amino acid sequence of PSMA-VH is shown in SEQ ID NO: 199
  • amino acid sequence of PSMA-VL is shown in SEQ ID NO: 202; or
  • amino acid sequence of the PSMA-VH is shown in SEQ ID NO: 188
  • amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 191; or
  • the antigen-binding molecule of any one of the preceding items wherein:
  • amino acid sequence of PSMA-VH is shown in SEQ ID NO: 204
  • amino acid sequence of PSMA-VL is shown in SEQ ID NO: 206.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • amino acid sequence of PSMA-VH is shown in SEQ ID NO: 197
  • amino acid sequence of PSMA-VL is shown in SEQ ID NO: 201.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • amino acid sequence of PSMA-VH is shown in SEQ ID NO: 199
  • amino acid sequence of PSMA-VL is shown in SEQ ID NO: 202.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding module that specifically binds CD28 or the antigen-binding module that specifically binds PSMA comprises a Titin chain and Obscurin capable of forming a dimer chain.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding module that specifically binds CD28 comprises a Titin chain and an Obscurin chain capable of forming a dimer.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding module that specifically binds PSMA includes a Titin chain and an Obscurin chain capable of forming a dimer.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 334 to SEQ ID NO: 352, and the Obscurin chain comprises an amino acid sequence selected from the group consisting of The amino acid sequence of the group consisting of SEQ ID NO: 353 to SEQ ID NO: 393.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 388.
  • the antigen-binding molecule of any one of the preceding items wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 350, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 388.
  • the antigen-binding molecule of any one of the preceding items wherein the antigen-binding molecule comprises an IgG Fc region.
  • the antigen-binding molecule of any one of the preceding items wherein the antigen-binding molecule comprises an IgG1 Fc region.
  • the antigen-binding molecule of any one of the preceding items wherein the antigen-binding molecule comprises an Fc region comprising one or more amino acid substitutions capable of reducing the binding of the Fc region to Fc ⁇ receptors.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region is a human IgG1 Fc region, and the amino acids at positions 234 and 235 are A, and the numbering basis is EU index.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, so The Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization of the Fc region.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule
  • the subunit includes an Fc region, the Fc region includes a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 has a convex structure according to the pestle and mortar technology, and the Fc2 has a pore structure according to the pestle and mortar technology.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, so
  • the Fc1 has a convex structure according to the pestle and mortar technology
  • the Fc2 has a pore structure according to the pestle and mortar technology
  • the amino acid of the Fc1 at position 366 is W
  • the amino acid of Fc2 at position 366 is S
  • the amino acid at position 368 is S.
  • the amino acid is A, and the amino acid at position 407 is V, and the numbering basis is the EU index.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, so
  • the Fc1 has a convex structure according to the pestle and mortar technology
  • the Fc2 has a pore structure according to the pestle and mortar technology, wherein the amino acid at the 354 position of the Fc1 is C and the amino acid at the 366 position is W; and the Fc2 is at 349
  • the amino acid at position 366 is S
  • the amino acid at position 368 is A
  • the amino acid at position 407 is V.
  • the numbering basis is the EU index.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein , the Fc1 includes the amino acid sequence of SEQ ID NO: 220; and the Fc2 includes the amino acid sequence of SEQ ID NO: 221 or 222.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein , the Fc1 includes the amino acid sequence of SEQ ID NO: 220; and the Fc2 includes the amino acid sequence of SEQ ID NO: 221.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an antigen-binding module that specifically binds CD28 and an antigen-binding module that specifically binds PSMA.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an antigen-binding module that specifically binds CD28 and an antigen-binding module that specifically binds PSMA, wherein:
  • the antigen-binding module that specifically binds to CD28 is a Fab
  • the antigen-binding module that specifically binds to PSMA is a replaced Fab that contains a Titin chain and an Obscurin chain capable of forming dimers.
  • the antigen-binding molecule as described in any one of the preceding items includes a first chain having a structure represented by formula (a) and a second chain having a structure represented by formula (b) , a third chain having the structure shown in formula (c) and a fourth chain having the structure shown in formula (d),
  • linker 1 and the linker 2 are the same or different peptide linkers; the structures shown in formulas (a), (b), (c) and (d) are arranged from the N end to the C end. of.
  • the antigen-binding molecule as described in any one of the preceding items includes a first chain having a structure represented by formula (a) and a second chain having a structure represented by formula (b) , a third chain having the structure shown in formula (c) and a fourth chain having the structure shown in formula (d),
  • the connector 1 and the connector 2 do not exist, that is, the connector 1 and the connector 2 are both bonds; as shown in formulas (a), (b), (c) and (d)
  • the structure is arranged from the N-terminus to the C-terminus.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204 sequence, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 206; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 33, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 34; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 65, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 71; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 78; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 31, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 32; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 sequence, and said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 50; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 206; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 206; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204 sequence, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 37
  • the CD28-VL includes the amino acid sequence of SEQ ID NO: 38
  • the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 sequence
  • said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, And the PSMA-VL contains the amino acid sequence of SEQ ID NO: 212.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, And the PSMA-VL contains the amino acid sequence of SEQ ID NO: 206.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 68, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 80; and the amino acid sequence of PSMA-VH is shown in SEQ ID NO: 204 is shown, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 206.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain and Obscurin chain can be selected from any Titin that can form dimers in Table 3-1 and Table 3-2 of the present disclosure. chain and Obscurin chain.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 350.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Obscurin chain comprises an amino acid sequence such as SEQ ID NO: 388.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the linker 1 and the linker 2 are both peptide linkers known in the art, as long as the antigen-binding molecule can exhibit the desired antigen binding active.
  • the peptide linker can be a flexible peptide having 1-50 or 3-20 amino acid residues.
  • the peptide linker is 3-15 amino acid residues in length.
  • the peptide linkers each independently have the structure (GGGGS)n, where n is 1, 2, or 3.
  • the sequences of linker 1 and linker 2 are GGGGS (SEQ ID NO: 223).
  • the antigen-binding molecule as described in any one of the preceding items, wherein said CH1 is IgG The CH1 sequence.
  • the CH1 is CH1 of IgG1.
  • the CH1 comprises the amino acid sequence of SEQ ID NO: 219.
  • the antigen-binding molecule as described in any one of the preceding items wherein said CL is the light chain constant region of an antibody. In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the CL is the light chain constant region of kappa or lamada. In some embodiments, the CL comprises the amino acid sequence of SEQ ID NO: 145.
  • a first strand as shown in SEQ ID NO:237, a second strand as shown in SEQ ID NO:239, a third strand as shown in SEQ ID NO:249 and a third strand as shown in SEQ ID NO:250 The fourth chain shown.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an antigen-binding module that specifically binds CD28 and an antigen-binding module that specifically binds PSMA, and the antigen-binding module that specifically binds PSMA
  • the antigen-binding module is a Fab; the antigen-binding module that specifically binds to CD28 is a replaced Fab, which contains a Titin chain and an Obscurin chain capable of forming dimers.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule includes a first chain having a structure shown in formula (e), a second chain having a structure shown in formula (f) chain, a third chain having the structure shown in formula (g) and a fourth chain having the structure shown in formula (h),
  • linker 3 and the linker 4 are the same or different peptide linkers; the structures shown in formulas (e), (f), (g) and (h) are arranged from the N-terminus to the C-terminus.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule includes a first chain having a structure shown in formula (e), a second chain having a structure shown in formula (f) chain, one a third chain having a structure represented by formula (g) and a fourth chain having a structure represented by formula (h),
  • the connector 3 and the connector 4 do not exist, that is, the connector 3 and the connector 4 are both bonds; the structures shown in formulas (e), (f), (g) and (h) are from Arranged from N-terminus to C-terminus.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204 sequence, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 94, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 sequence, and said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 37, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 38; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 sequence, and said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL Comprising the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH comprising the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 129, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 138; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 206; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, And the PSMA-VL contains the amino acid sequence of SEQ ID NO: 208.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, And the PSMA-VL contains the amino acid sequence of SEQ ID NO: 201.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, And the PSMA-VL contains the amino acid sequence of SEQ ID NO: 206.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 95, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 101; and the amino acid sequence of PSMA-VH is shown in SEQ ID NO: 197 is shown, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 201.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 95, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 101; and the amino acid sequence of PSMA-VH is shown in SEQ ID NO: 204 is shown, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 206.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain and The Obscurin chain can be selected from any Titin chain and Obscurin chain that can form dimers in Table 3-1 and Table 3-2 in this disclosure.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 350.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Obscurin chain comprises an amino acid sequence such as SEQ ID NO: 388.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the linker 3 and the linker 4 are both peptide linkers known in the art, as long as the antigen-binding molecule can exhibit the desired antigen binding active.
  • the peptide linker can be a flexible peptide containing 1-50 or 3-20 amino acid residues.
  • the peptide linker is 3-15 amino acid residues in length.
  • the peptide linkers each independently have the structure (GGGGS)n, where n is 1, 2, or 3.
  • the sequences of linker 3 and linker 4 are GGGGS (SEQ ID NO: 223).
  • the antigen-binding molecule as described in any one of the preceding items, wherein the CH1 is the CH1 sequence of IgG. In some embodiments, the CH1 is CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 219.
  • the antigen-binding molecule as described in any one of the preceding items wherein said CL is the light chain constant region of an antibody. In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the CL is the light chain constant region of kappa or lamada. In some embodiments, the CL comprises the amino acid sequence of SEQ ID NO: 145.
  • a first strand as shown in SEQ ID NO:261, a second strand as shown in SEQ ID NO:262, a third strand as shown in SEQ ID NO:272 and a third strand as shown in SEQ ID NO:273 The fourth chain shown.
  • a first strand as shown in SEQ ID NO: 261, a second strand as shown in SEQ ID NO: 262, a third strand as shown in SEQ ID NO: 274 and a third strand as shown in SEQ ID NO: 275 The fourth chain shown.
  • the present disclosure also provides an anti-CD28 antibody capable of specifically binding to CD28, the anti-CD28 antibody comprising a heavy chain variable region CD28-VH and a light chain variable region CD28-VL, wherein:
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 35
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL contain CD28 in SEQ ID NO: 36, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115 respectively -amino acid sequences of LCDR1, CD28-LCDR2 and CD28-LCDR3, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL contain CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 34, 73, 74, 75, 76, 77, 78, 79 or 80 respectively the amino acid sequence, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 37, 129 or 132
  • CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in the CD28-VL respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 38, 139, 141, 142 or 143, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 32, 46, 47, 48 or 49.
  • the anti-CD28 antibody as described above, wherein:
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 35
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 36, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 80, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33, and the CD28-VL in the CD28-VL.
  • CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 34, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 37
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 38, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 32;
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, and the CD28-VL in the CD28-VL.
  • CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 52.
  • the CD28-HCDR1, CD28-HCDR2, CD28-HCDR3, CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 are defined according to Kabat, IMGT, Chothia, AbM or Contact numbering rules.
  • the anti-CD28 antibody as described above, wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 24 CD28-LCDR3; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15.
  • HCDR3 has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, comprising the amino acid sequence of SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62
  • the sequence of CD28-LCDR2 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 18; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25 or 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 116 and the amino acid sequence comprising SEQ ID NO: 27 CD28-HCDR3 of the sequence, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28- comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 LCDR2 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42 amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or 43, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or 43. ID NO: 12 amino acid sequence of CD28-LCDR3.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, A CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 and a CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 30 amino acid sequence of CD28-LCDR3.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23 and CD28- comprising the amino acid sequence of SEQ ID NO: 24 LCDR3; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15.
  • HCDR3 has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 62 and CD28- comprising the amino acid sequence of SEQ ID NO: 18 LCDR3; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 27.
  • HCDR3 has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29 and CD28- comprising the amino acid sequence of SEQ ID NO: 30 LCDR3; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28- comprising the amino acid sequence of SEQ ID NO: 9 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 12 LCDR3.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 22, such as SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 CD28-LCDR2 is shown and as CD28-LCDR3 shown in SEQ ID NO: 24; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 16, CD28-LCDR2 as shown in SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and CD28-LCDR3 shown in SEQ ID NO: 18; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25 or 117, CD28-HCDR2 as shown in SEQ ID NO: 26 or 116, and CD28-HCDR1 as shown in SEQ ID NO: 27 HCDR3, and the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 28, CD28-LCDR2 as set forth in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 as set forth in SEQ ID NO: CD28-LCDR3 shown in 30; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 7, CD28-HCDR2 as shown in SEQ ID NO: 8 and CD28-HCDR2 as shown in SEQ ID NO: 9, 39, 40, 41 or 42 CD28-HCDR3 as shown, and the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 10, CD28-LCDR2 as shown in SEQ ID NO: 11 or 43, and CD28-LCDR2 as shown in SEQ ID NO: 12 CD28-LCDR3.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116 and amino acid sequence comprising SEQ ID NO: 27 CD28-HCDR3 with amino acid sequence
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 CD28-LCDR2 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO:30.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42.
  • the amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 12
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42.
  • the amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 12 Amino acid sequence of CD28-LCDR3.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and
  • the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 22, CD28-LCDR2 as set forth in SEQ ID NO: 23, and CD28-LCDR3 as set forth in SEQ ID NO: 24; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and
  • the CD28-VL is CD28-LCDR1 set forth in SEQ ID NO: 16, CD28-LCDR2 set forth in SEQ ID NO: 62, and CD28-LCDR3 set forth in SEQ ID NO: 18; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25, CD28-HCDR2 as shown in SEQ ID NO: 26 and CD28-HCDR3 as shown in SEQ ID NO: 27, and
  • the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 28, CD28-LCDR2 as set forth in SEQ ID NO: 29, and CD28-LCDR3 as set forth in SEQ ID NO: 30; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO:7, CD28-HCDR2 as shown in SEQ ID NO:8 and CD28-HCDR3 as shown in SEQ ID NO:9, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 10, CD28-LCDR2 as shown in SEQ ID NO: 43, and CD28-LCDR3 as shown in SEQ ID NO: 12.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and the CD28- VL includes CD28-LCDR1 as shown in SEQ ID NO:22, CD28-LCDR2 as shown in SEQ ID NO:23, and CD28-LCDR3 as shown in SEQ ID NO:24.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and the CD28- VL is CD28-LCDR1 as shown in SEQ ID NO: 16, CD28-LCDR2 as shown in SEQ ID NO: 62, and CD28-LCDR3 as shown in SEQ ID NO: 18.
  • the anti-CD28 antibody of any one of the preceding items are defined according to the Kabat numbering rule.
  • the anti-CD28 antibody as described in any one of the preceding items wherein the CD28-VH and CD28-VL are humanized and both comprise the FR region of a human antibody.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 26A, 29L, 69L, 71V, 78A and 93S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 with the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 with the amino acid sequence of SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 and a CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and the FR region of the CD28-VL comprises one or more amino acid substitutions selected from the group consisting
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15.
  • the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 69L, 71V, 73K and 94S; and the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 with the amino acid sequence of SEQ ID NO: 16, CD28-LCDR2 with the amino acid sequence of SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and the amino acid sequence of SEQ ID NO: 18 CD28-LCDR3, and the FR region of CD28-VL contains one or more amino acid substitutions selected from the group consisting of 43S and 70K; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25 or 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 116 and the amino acid sequence comprising SEQ ID NO: 27
  • the sequence of CD28-HCDR3, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 71V and 78A
  • the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 having the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 having the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 and CD28-LCDR3 having the amino acid sequence of SEQ ID NO: 30, and said
  • the FR region of CD28-VL contains one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44V and 73L; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42, and the FR region of the CD28-VH comprises a FR region selected from 1E, One or more amino acid substitutions in the group consisting of 69L, 71V, 73K, 78A and 94S; and the CD28-VL has: a CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 11 or CD28-LCDR2 with the amino acid sequence of SEQ ID NO: 12 and CD28-LCDR3 with the amino acid sequence of SEQ ID NO: 12; and the FR region of CD28-VL includes one or more amino acid substitutions selected from the group consisting of 43S and 73
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 26A, 29L, 69L, 71V, 78A and 93S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 23, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 24, and the FR region of CD28-VL includes the selected One or more amino acid substitutions from the group consisting of 41D, 42G, 43T, 44I and 71Y; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15.
  • the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 69L, 71V, 73K and 94S; and the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 62, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 62, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 18, and the FR region of the CD28-VL contains a protein selected from the group consisting of 43S and one or more amino acid substitutions in the group consisting of 70K; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 27.
  • CD28-VL has: the amino acid sequence comprising SEQ ID NO: 28 CD28-LCDR1, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30, and the FR region of the CD28-VL comprises 41D, 42G, 43T One or more amino acid substitutions in the group consisting of , 44V and 73L; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28- comprising the amino acid sequence of SEQ ID NO: 9 HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S; and the CD28-VL has: comprising SEQ CD28-LCDR1 with the amino acid sequence of ID NO: 10, CD28-LCDR2 with the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR3 with the amino acid sequence of SEQ ID NO: 12; and the FR region of CD28-VL includes One or more amino acid substitutions selected from the group consisting of 43S and 73F.
  • the anti-CD28 antibody as described in any one of the preceding items binds to human CD28 with a KD of less than 2 ⁇ 10 -8 M, 8 ⁇ 10 -9 M or 5 ⁇ 10 -9 M at 25°C. , the KD is measured by surface plasmon resonance method.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 98 or a variant thereof, the variant is included in the FR region of SEQ ID NO: 98 and the FR region of the CD28-VH includes a sequence selected from 26A , 29L, 69L, 71V, 78A and 93S, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 102 or a variant thereof, the variant being in SEQ ID NO.
  • the FR region of NO: 102 contains one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44I and 71Y; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 69 or a variant thereof, wherein the variant contains a sequence selected from the group consisting of 28S, 69L, 71V, 73K and 94S in the FR region of SEQ ID NO: 69 One or more amino acid substitutions in the group, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70 or a variant thereof, the variant being in the FR region of SEQ ID NO: 70 and including 43S and 70K One or more amino acid substitutions in the group consisting of; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 133 or a variant thereof, wherein the variant is one selected from the group consisting of 25P, 71V and 78A in the FR region of SEQ ID NO: 133 Or multiple amino acid substitutions, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 137 or a variant thereof, the variant is in the FR region of SEQ ID NO: 137.
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 53 or a variant thereof, wherein the variant contains a FR region selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S of SEQ ID NO: 53 consisting of one or more amino acid substitutions in the group, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or a variant thereof, the variant being in the FR region of SEQ ID NO: 50 and comprising 43S selected from the group consisting of One or more amino acid substitutions in the group consisting of 73F; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 53 or a variant thereof, wherein the variant is a group selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S in the FR region of SEQ ID NO: 53.
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52 or a variant thereof, wherein the variant is composed of a FR region selected from 43S and 73F of SEQ ID NO: 52
  • One or more amino acids in the group are substituted.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 35, 94, 96, 97 or 98
  • the CD28-VL comprises SEQ ID NO: 101, 36, 99, 100, 102, 103 ,104,105
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, 33, 63, 64, 65, 66, 67 or 69
  • the CD28-VL comprises SEQ ID NO: 80, 34, 70, 71 , 72, 73, 74, 75, 76, 77, 78 or 79 amino acid sequence
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, 37, 123, 124, 125, 127, 128, 129, 130, 131, 132 or 133
  • the CD28-VL comprises SEQ ID NO. : the amino acid sequence of 134, 38, 135, 136, 137, 138, 139, 140, 141, 142 or 143; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, 31, 45, 46, 47, 48, 49 or 53
  • the CD28-VL comprises SEQ ID NO: 52, 32, 50 or 51 amino acid sequence.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98
  • the CD28-VL comprises SEQ ID NO: 101, 99, 100, 102, 103, 104, 105 , the amino acid sequence of 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, 63, 64, 65, 66, 67 or 69
  • the CD28-VL comprises SEQ ID NO: 80, 70, 71, 72, 73 , 74, 75, 76, 77, 78 or 79 amino acid sequence
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, 123, 124, 125, 127, 128, 129, 130, 131, 132 or 133
  • the CD28-VL comprises SEQ ID NO: 134 , 135, 136, 137, 138, 139, 140, 141, 142 or 143 amino acid sequence
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, 45, 46, 47, 48 or 49
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52, 50, 51 or 53.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 36;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 33
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 34;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 37
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 38;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 31
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 32.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101, 99, 100 or 102, or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 94
  • the CD28-VL includes SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, the amino acid sequence of 112, 113, 114 or 115, or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95
  • the CD28-VL includes SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, the amino acid sequence of 112, 113, 114 or 115, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 96
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99 or 100, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 97
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99, 100 or 101, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 63
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70 or 71
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 64
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71 or 72, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 65
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71 or 72, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 66
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71, 72, 74, 75, 76 or 79, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 67
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 74, 75, 76 or 79, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79 or 80, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 123
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135 or 136, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 124, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 135; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 125
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134 or 135, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135, 136, 137, 138 or 139, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 127
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 137, 138 or 139; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 128, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 137, 138, 139 or 142; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 129
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 138, 139, 140, 141, 142 or 143, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 130
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 131
  • the CD28-VL comprises The amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50, 51 or 52, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 45
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 46
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 52, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 47
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 48
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 80
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 95, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 101 shown.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 68, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 80 shown.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 126, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 134 shown.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 44, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 52 shown.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody comprises a heavy chain constant region and a light chain constant region.
  • the anti-CD28 antibody of any one of the preceding items wherein the heavy chain constant region is a human IgGl constant region.
  • the anti-CD28 antibody as described in any one of the preceding items wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 144.
  • the anti-CD28 antibody as described in any one of the preceding items wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO: 145.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody comprises a heavy chain and a light chain, wherein:
  • the heavy chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% sequence identity to an amino acid sequence
  • the light chain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%
  • An amino acid sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical; or
  • the heavy chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% sequence identity to an amino acid sequence
  • the light chain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%
  • An amino acid sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical; or
  • the heavy chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% sequence identity to an amino acid sequence
  • the light chain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%
  • An amino acid sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical; or
  • the heavy chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% sequence identity to an amino acid sequence
  • the light chain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, Amino acid sequences with 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody comprises a heavy chain and a light chain, wherein:
  • the heavy chain includes the amino acid sequence of SEQ ID NO: 146, and the light chain includes the amino acid sequence of SEQ ID NO: 147;
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 148, and the light chain comprises the amino acid sequence of SEQ ID NO: 149; or
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 150, and the light chain comprises the amino acid sequence of SEQ ID NO: 151; or
  • the heavy chain includes the amino acid sequence of SEQ ID NO:152, and the light chain includes the amino acid sequence of SEQ ID NO:153.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 150, and the light chain The chain contains the amino acid sequence of SEQ ID NO: 151.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 148, and the light chain The chain contains the amino acid sequence of SEQ ID NO: 149.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody is an antibody fragment, wherein the antibody fragment is Fab, Fab', F(ab')2, Fd, Fv , scFv, dsFv or dAb.
  • the anti-CD28 antibody of any one of the preceding items wherein the anti-CD28 antibody is a bispecific antibody.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody is a bispecific antibody, and the bispecific antibody specifically binds to CD28 and PSMA.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (e.g., a therapeutically effective amount) the antigen-binding molecule of any of the foregoing or the anti-CD28 antibody of any of the foregoing, and a or more pharmaceutically acceptable carriers, diluents, buffers or excipients.
  • the pharmaceutical composition further includes at least one (eg, one) second therapeutic agent.
  • the second therapeutic agent is any agent that is advantageously combined with a PSMA/CD28 bispecific antigen-binding molecule (including antineoplastic agents, radiotherapy, antibody drug conjugates, bispecific antibodies, and antineoplastic agent-conjugated bispecific antibodies, immune checkpoint inhibitors, or combinations thereof).
  • the second therapeutic agent is an antibody capable of specifically binding CD3; preferably, the second therapeutic agent is a bispecific antibody capable of specifically binding CD3; more preferably, the second therapeutic agent is a bispecific antibody capable of specifically binding CD3.
  • the therapeutic agent is a bispecific antibody that specifically binds to CD3 and tumor cell surface antigen (TAA); most preferably, the second therapeutic agent is a bispecific antibody that specifically binds to CD3 and PSMA.
  • the second therapeutic agent is an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor targets PD-1 or CTLA4; preferably, the immune checkpoint inhibitor is an anti- PD-1 antibodies.
  • the present disclosure also provides an isolated nucleic acid encoding the antigen-binding molecule of any of the foregoing or the anti-CD28 antibody of any of the foregoing.
  • the present disclosure also provides a vector comprising the aforementioned isolated nucleic acid.
  • the present disclosure also provides a host cell comprising the aforementioned vector.
  • the present disclosure also provides a method of treating a disease, the method comprising administering (e.g., a therapeutically effective amount) to a subject the antigen-binding molecule of any of the foregoing or the antigen-binding molecule of any of the foregoing.
  • administering e.g., a therapeutically effective amount
  • Anti-CD28 antibody or a composition as described in any one of the preceding items.
  • the present disclosure also provides the antigen-binding molecule of any of the foregoing or any of the foregoing.
  • the present disclosure also provides the antigen-binding molecule of any of the foregoing, the anti-CD28 antibody of any of the foregoing, or the composition of any of the foregoing for use as a medicament.
  • the medicament is used to treat disease.
  • the disease of any of the preceding items is a tumor.
  • the tumor of any one of the preceding items is prostate cancer, renal cancer, urothelial cancer, breast cancer, bladder cancer, primary liver cancer, pancreatic cancer, melanoma, glioblastoma (e.g.
  • Glioblastoma multiforme osteosarcoma, ovarian cancer, esophageal cancer, cervical squamous cell carcinoma, lung cancer (non-small cell lung cancer, small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma), colorectal cancer (including colon and rectal cancer), endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain cancer, gastroesophageal cancer (gastroesophageal adenocarcinoma), metastatic liver cancer, multiple myeloma, lymphoma , acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) or B-cell lymphoma.
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • PSMA is expressed by cells of a tumor as described above or by vascular endothelial cells proximate the tumor.
  • the tumor as described above is a solid tumor.
  • the tumor as described above is a solid tumor
  • the solid tumor is prostate cancer, renal cancer, urothelial cancer, breast cancer, bladder cancer, primary liver cancer, pancreatic cancer, melanoma, glaucoma, Blastoma (such as glioblastoma multiforme), osteosarcoma, ovarian cancer, esophageal cancer, cervical squamous cell carcinoma, lung cancer (non-small cell lung cancer, small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma cancer), colorectal cancer (including colon cancer and rectal cancer), endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain cancer, gastroesophageal cancer (gastroesophageal adenocarcinoma), liver metastatic cancer.
  • prostate cancer renal cancer, urothelial cancer, breast cancer, bladder cancer, primary liver cancer, pancreatic cancer, melanoma, glaucoma, Blastoma (such
  • the tumor as described above is prostate cancer.
  • the tumor as described above is a non-solid tumor.
  • the tumor as described above is a non-solid tumor, the non-solid tumor being multiple myeloma, lymphoma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia Cellular leukemia (CLL) or B-cell lymphoma.
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia Cellular leukemia
  • B-cell lymphoma B-cell lymphoma
  • the prostate cancer as described above is refractory prostate cancer, prostatic intraepithelial neoplasia, androgen-independent prostate cancer, malignant prostate cancer, recurrent prostate cancer, or castration-resistant prostate cancer.
  • the aforementioned disease is a disease associated with PSMA; in some embodiments, the aforementioned disease is a disease expressing PSMA.
  • the method of treating a disease as described above further includes using a second therapeutic agent.
  • the second therapeutic agent includes an anti-tumor agent, radiation therapy, antibody drug conjugates, bispecific antibodies, bispecific antibodies conjugated to an anti-tumor agent, immune checkpoint inhibitors or combination thereof.
  • the second therapeutic agent is an antibody capable of specifically binding CD3; preferably, the second therapeutic agent is a bispecific antibody capable of specifically binding CD3; more preferably, The second therapeutic agent is a bispecific antibody that can specifically bind to CD3 and tumor cell surface antigen (TAA); most preferably, the second therapeutic agent is a bispecific antibody that can specifically bind to CD3 and PSMA.
  • the second therapeutic agent is an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor targets PD-1 or CTLA4; preferably, the immune checkpoint inhibitor is an anti- PD-1 antibodies.
  • the second therapeutic agent of any one of the preceding and the antigen-binding molecule of any of this disclosure are administered simultaneously, sequentially, or separately.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising at least one antigen-binding moiety that specifically binds PSMA and at least one antigen-binding moiety that specifically binds CD3.
  • Antigen binding module is a bispecific antibody that specifically binds PSMA and CD3, comprising at least one antigen-binding moiety that specifically binds PSMA and at least one antigen-binding moiety that specifically binds CD3.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising at least one antigen-binding moiety that specifically binds PSMA and at least one antigen-binding moiety that specifically binds CD3.
  • Antigen-binding module wherein the antigen-binding module that specifically binds to PSMA binds to a different epitope than the antigen-binding module that specifically binds to CD28 and PSMA in the antigen-binding molecule that specifically binds to CD28 and PSMA.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising at least one antigen-binding moiety that specifically binds PSMA and at least one antigen-binding moiety that specifically binds CD3.
  • Antigen-binding module, the antigen-binding module that specifically binds to PSMA includes the heavy chain variable region PSMA-VH and the light chain variable region PSMA-VL, and the antigen-binding module that specifically binds to CD3 includes the heavy chain variable region CD3 -VH and light chain variable region CD3-VL.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 164, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 165, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 166 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 167, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 168, and PSMA comprising the amino acid sequence of SEQ ID NO: 169 -LCDR3; or
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 170, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 171, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 172 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 173, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 174, and PSMA comprising the amino acid sequence of SEQ ID NO: 175 -LCDR3; or
  • the PSMA-VH has: comprising the amino acid sequence of SEQ ID NO: 158 PSMA-HCDR1, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 159, and PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 160; and said PSMA-VL has: comprising the amino acid sequence of SEQ ID NO: 161 PSMA-LCDR1, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 162, and PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 163; or
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 176, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 177, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 178 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 179, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 180, and PSMA comprising the amino acid sequence of SEQ ID NO: 181 -LCDR3; or
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 301, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 302, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 303 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 304, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 305, and PSMA comprising the amino acid sequence of SEQ ID NO: 306 -LCDR3.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH has: PSMA-HCDR1 including the amino acid sequence of SEQ ID NO: 170, PSMA-HCDR2 including the amino acid sequence of SEQ ID NO: 171, and PSMA-HCDR3 including the amino acid sequence of SEQ ID NO: 172; And the PSMA-VL has: PSMA-LCDR1 including the amino acid sequence of SEQ ID NO: 173, PSMA-LCDR2 including the amino acid sequence of SEQ ID NO: 174, and PSMA-LCDR3 including the amino acid sequence of SEQ ID NO: 175 .
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH has: PSMA-HCDR1 including the amino acid sequence of SEQ ID NO: 164, PSMA-HCDR2 including the amino acid sequence of SEQ ID NO: 165, and PSMA-HCDR3 including the amino acid sequence of SEQ ID NO: 166; And the PSMA-VL has: PSMA-LCDR1 including the amino acid sequence of SEQ ID NO: 167, PSMA-LCDR2 including the amino acid sequence of SEQ ID NO: 168, and PSMA-LCDR3 including the amino acid sequence of SEQ ID NO: 169
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH comprises PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and
  • the PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 167, as shown in SEQ ID NO: PSMA-LCDR2 as shown in 168, and PSMA-LCDR3 as shown in SEQ ID NO: 169; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and Said PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 173, PSMA-LCDR2 as shown in SEQ ID NO: 174, and PSMA-LCDR3 as shown in SEQ ID NO: 175; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 158, PSMA-HCDR2 as shown in SEQ ID NO: 159, and PSMA-HCDR3 as shown in SEQ ID NO: 160; and Said PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 161, PSMA-LCDR2 as shown in SEQ ID NO: 162, and PSMA-LCDR3 as shown in SEQ ID NO: 163; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 176, PSMA-HCDR2 as shown in SEQ ID NO: 177, and PSMA-HCDR3 as shown in SEQ ID NO: 178; and Said PSMA-VL includes PSMA-LCDR1 as set forth in SEQ ID NO: 179, PSMA-LCDR2 as set forth in SEQ ID NO: 180, and PSMA-LCDR3 as set forth in SEQ ID NO: 181; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 301, PSMA-HCDR2 as shown in SEQ ID NO: 302, and PSMA-HCDR3 as shown in SEQ ID NO: 303; and
  • the PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 304, PSMA-LCDR2 as shown in SEQ ID NO: 305, and PSMA-LCDR3 as shown in SEQ ID NO: 306.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and the PSMA -VL contains PSMA-LCDR1 as shown in SEQ ID NO: 173, PSMA-LCDR2 as shown in SEQ ID NO: 174, and PSMA-LCDR3 as shown in SEQ ID NO: 175.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and the PSMA -VL contains PSMA-LCDR1 as shown in SEQ ID NO: 167, PSMA-LCDR2 as shown in SEQ ID NO: 168, and PSMA-LCDR3 as shown in SEQ ID NO: 169.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, 199, 332, 194, 195, 196 or 198
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201, 202, 333 or 200 ;
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, 184 or 203
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206, 185 or 205; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188, 182, 189 or 190
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191, 183, 192 or 193; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 207 or 186
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 208, 187, 209 or 210; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 313, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 314.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201;
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191;
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 202; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 207
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 208.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein said PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, and said PSMA -VL contains the amino acid sequence of SEQ ID NO: 206.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein said PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, and said PSMA -VL contains the amino acid sequence of SEQ ID NO:201.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein said PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199, and said PSMA -VL contains the amino acid sequence of SEQ ID NO:202.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and the light chain variable region CD3-VL, where:
  • the CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 283, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 284, and CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 285 HCDR3; and the CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 286, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 287, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 288; or
  • the CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 307, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 308, and CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 309 HCDR3; and the CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 310, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 311, and CD3 comprising the amino acid sequence of SEQ ID NO: 312 -LCDR3.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and the light chain variable region CD3-VL, where:
  • the CD3-VH has: CD3-HCDR1 including the amino acid sequence of SEQ ID NO: 283, CD3-HCDR2 including the amino acid sequence of SEQ ID NO: 284, and CD3-HCDR3 including the amino acid sequence of SEQ ID NO: 285; And the CD3-VL has: CD3-LCDR1 including the amino acid sequence of SEQ ID NO: 286, CD3-LCDR2 including the amino acid sequence of SEQ ID NO: 287, and CD3-LCDR3 including the amino acid sequence of SEQ ID NO: 288 .
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and the light chain variable region CD3-VL, where:
  • the CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 283, CD3-HCDR2 as shown in SEQ ID NO: 284, and CD3-HCDR3 as shown in SEQ ID NO: 285; and
  • the CD3-VL includes CD3-LCDR1 as set forth in SEQ ID NO: 286, CD3-LCDR2 as set forth in SEQ ID NO: 287, and CD3-LCDR3 as set forth in SEQ ID NO: 288; or
  • the CD3-VH includes CD3-HCDR1 as set forth in SEQ ID NO: 307, CD3-HCDR2 as set forth in SEQ ID NO: 308, and CD3-HCDR3 as set forth in SEQ ID NO: 309; and
  • the CD3-VL includes CD3-LCDR1 as shown in SEQ ID NO:310, CD3-LCDR2 as shown in SEQ ID NO:311, and CD3-LCDR3 as shown in SEQ ID NO:312.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and the light chain variable region CD3-VL, where:
  • the CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 283, CD3-HCDR2 as shown in SEQ ID NO: 284, and CD3-HCDR3 as shown in SEQ ID NO: 285; and the CD3 -VL contains CD3-LCDR1 as shown in SEQ ID NO:286, CD3-LCDR2 as shown in SEQ ID NO:287, and CD3-LCDR3 as shown in SEQ ID NO:288.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, said CD3-VH comprising the amino acid sequence of SEQ ID NO: 289, and said CD3- VL contains the amino acid sequence of SEQ ID NO: 290; or
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO: 315
  • the CD3-VL comprises SEQ Amino acid sequence of ID NO:316.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and the PSMA - VL contains PSMA-LCDR1 as set forth in SEQ ID NO: 173, PSMA-LCDR2 as set forth in SEQ ID NO: 174, and PSMA-LCDR3 as set forth in SEQ ID NO: 175; and
  • the CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 283, CD3-HCDR2 as shown in SEQ ID NO: 284, and CD3-HCDR3 as shown in SEQ ID NO: 285; and the CD3 -VL contains CD3-LCDR1 as shown in SEQ ID NO:286, CD3-LCDR2 as shown in SEQ ID NO:287, and CD3-LCDR3 as shown in SEQ ID NO:288.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and the PSMA -VL contains PSMA-LCDR1 as set forth in SEQ ID NO: 167, PSMA-LCDR2 as set forth in SEQ ID NO: 168, and PSMA-LCDR3 as set forth in SEQ ID NO: 169; and
  • the CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 283, CD3-HCDR2 as shown in SEQ ID NO: 284, and CD3-HCDR3 as shown in SEQ ID NO: 285; and the CD3 -VL contains CD3-LCDR1 as shown in SEQ ID NO:286, CD3-LCDR2 as shown in SEQ ID NO:287, and CD3-LCDR3 as shown in SEQ ID NO:288.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 301, PSMA-HCDR2 as shown in SEQ ID NO: 302, and PSMA-HCDR3 as shown in SEQ ID NO: 303; and the PSMA -VL contains PSMA-LCDR1 as shown in SEQ ID NO:304, PSMA-LCDR2 as shown in SEQ ID NO:305, and PSMA-LCDR3 as shown in SEQ ID NO:306; and
  • the CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 307, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 308, and CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 309; And the CD3-VL has: CD3-LCDR1 including the amino acid sequence of SEQ ID NO: 310, CD3-LCDR2 including the amino acid sequence of SEQ ID NO: 311, and CD3-LCDR3 including the amino acid sequence of SEQ ID NO: 312 .
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206;
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO: 289
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO: 290.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 201;
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO: 289
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO: 290.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes the amino acid sequence of SEQ ID NO: 199, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 202; and
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO: 289
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO: 290.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes the amino acid sequence of SEQ ID NO: 313, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 314; and
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO: 315
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO: 316.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising an Fc region.
  • the Fc region is an IgG Fc region.
  • the Fc region is an IgGl Fc region.
  • the Fc region contains one or more amino acid substitutions that reduce binding of the Fc region to Fc ⁇ receptors.
  • the Fc region is a human IgGl Fc region, and the amino acids at positions 234 and 235 are A, numbered according to the EU index.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising an Fc region comprising a first subunit Fcl capable of associating with each other and a second subunit Fc2, each of said Fc1 and Fc2 independently having one or more amino acid substitutions that reduce homodimerization of the Fc region.
  • the Fc1 has a convex structure according to the pestle and mortar technology
  • the Fc2 has a pore structure according to the pestle and mortar technology.
  • the amino acid at position 366 of Fc1 is W; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbering according to EU index.
  • the Fc1 amino acid at position 354 is C and the amino acid at position 366 is W; and the Fc2 amino acid at position 349 is C, the amino acid at position 366 is S, and the amino acid at position 368 is W is A, and the amino acid at position 407 is V, and the numbering basis is the EU index.
  • the Fcl comprises The amino acid sequence of SEQ ID NO: 220; the Fc2 includes the amino acid sequence of SEQ ID NO: 221.
  • the Fc1 comprises the amino acid sequence of SEQ ID NO:220; the Fc2 comprises the amino acid sequence of SEQ ID NO:222.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising an antigen-binding moiety that specifically binds PSMA and an antigen-binding moiety that specifically binds CD3.
  • module, the antigen-binding module that specifically binds to PSMA and the antigen-binding module that specifically binds to CD3 are both scFv.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (a) and a first chain having a structure represented by formula ( b) The second strand of the structure shown,
  • linker 6, linker 7, linker 8 and/or linker 9 are the same or different peptide linkers.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (a) and a first chain having a structure represented by formula ( b) The second strand of the structure shown,
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197; and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201; and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 289; and The CD3-V L includes the amino acid sequence of SEQ ID NO: 290.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (a) and a first chain having a structure represented by formula ( b) The second strand of the structure shown,
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199; and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201; and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 289 Amino acid sequence; and said CD3-V L comprises the amino acid sequence of SEQ ID NO: 290.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (a) and a first chain having a structure represented by formula ( b) The second strand of the structure shown,
  • the structures shown in formulas (a) and (b) are arranged from the N end to the C end.
  • the linker 6 and the linker 8 are peptide linkers with the same sequence; the linker 7 and the linker 9 have different sequences. Peptide linker.
  • the second therapeutic agent as described in any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, and the linker 6, linker 7, linker 8, and linker 9 can be any Suitable peptide linkers are as long as the antigen-binding molecule can exhibit the desired antigen-binding activity.
  • the peptide linker can be a flexible peptide containing 1-50 or 3-20 amino acid residues.
  • the peptide linkers each independently have the structure of L 1 -(GGGGS)nL 2 , wherein L 1 is a bond, S, GS, GGS (SEQ ID NO: 399) or GGGS (SEQ ID NO: 400), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is key, G, GG, GGG (SEQ ID NO: 295) or GGGG (SEQ ID NO: 256), and the peptide linker is not a bond.
  • the peptide linker is 3-15 amino acid residues in length.
  • the second therapeutic agent as described in any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein said linker 6 and linker 8 comprise the amino acid sequence of SEQ ID NO: 293 (GGGGGSGGGGSGGGGS).
  • the linker 7 includes the amino acid sequence of SEQ ID NO: 294 (SGGGGS).
  • the linker 9 includes the amino acid sequence of SEQ ID NO: 295 (GGG).
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein said Fc1 comprises the amino acid sequence of SEQ ID NO: 220.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the Fc2 comprises the amino acid sequence of SEQ ID NO: 221.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the bispecific antibody that specifically binds PSMA and CD3 has: a bar comprising SEQ ID The first strand contains the amino acid sequence of SEQ ID NO: 296 and the second strand contains the amino acid sequence of SEQ ID NO: 220.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the bispecific antibody that specifically binds PSMA and CD3 has:
  • a first strand comprising the amino acid sequence of SEQ ID NO: 297 and a second strand comprising the amino acid sequence of SEQ ID NO: 220.
  • the second therapeutic agent of any one of the preceding items specifically binds PSMA and Bispecific antibodies to CD3, which comprise an antigen-binding module that specifically binds PSMA and an antigen-binding module that specifically binds CD3, and the antigen-binding module that specifically binds PSMA or the antigen-binding module that specifically binds CD3 includes an antigen-binding module that specifically binds to PSMA. Titin chain and Obscurin chain forming a dimer.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising an antigen-binding moiety that specifically binds PSMA and an antigen-binding moiety that specifically binds CD3.
  • the antigen-binding module that specifically binds to PSMA is a Fab
  • the antigen-binding module that specifically binds to CD3 is a replaced Fab, which includes a Titin chain and an Obscurin chain capable of forming dimers.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the antigen-binding moiety that specifically binds CD3 is a Fab; said specifically binds PSMA
  • the antigen-binding module is a replaced Fab containing a Titin chain and an Obscurin chain capable of forming dimers.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (c), a first chain having a structure represented by formula (c), d) a second chain having the structure shown in formula (e), a third chain having the structure shown in formula (e) and a fourth chain having the structure shown in formula (f),
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (c), a first chain having a structure represented by formula (c), d) a second chain having the structure shown in formula (e), a third chain having the structure shown in formula (e) and a fourth chain having the structure shown in formula (f),
  • the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204; and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 206; and the CD3-VH includes the amino acid sequence of SEQ ID NO: 289; and The CD3-VL includes the amino acid sequence of SEQ ID NO: 290.
  • the second therapeutic agent of any one of the preceding items specifically binds PSMA and A bispecific antibody to CD3, wherein the Titin chain and Obscurin chain are any Titin chain and Obscurin chain in Table 3-1 and Table 3-2 of the present disclosure that can form dimers.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 350.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Obscurin chain comprises an amino acid sequence such as SEQ ID NO: 388.
  • the second therapeutic agent as described in any one of the preceding is a bispecific antibody that specifically binds PSMA and CD3, wherein the linker 10 and linker 11 can be any suitable peptide linkage as long as the antigen-binding molecule can exhibit the desired antigen-binding activity.
  • the peptide linker can be a flexible peptide containing 1-50 or 3-20 amino acid residues.
  • the peptide linkers each independently have the structure of L 1 -(GGGGS)nL 2 , wherein L 1 is a bond, S, GS, GGS (SEQ ID NO: 399) or GGGS (SEQ ID NO: 400), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is key, G, GG, GGG (SEQ ID NO: 295) or GGGG (SEQ ID NO: 256), and the peptide linker is not a bond.
  • the peptide linker is 3-15 amino acid residues in length.
  • the linker 10 and the linker 11 comprise the amino acid sequence set forth in SEQ ID NO:223.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the CH1 is the CH1 sequence of an IgG.
  • the CH1 is CH1 of IgG1.
  • the CH1 comprises the amino acid sequence of SEQ ID NO: 219.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the CL is the light chain constant region of kappa or lamada.
  • the CL comprises the amino acid sequence of SEQ ID NO: 145.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, having: a first chain comprising the amino acid sequence of SEQ ID NO: 298, a first chain comprising A second strand containing the amino acid sequence of SEQ ID NO: 275, a third strand containing the amino acid sequence of SEQ ID NO: 291 and a fourth strand containing the amino acid sequence of SEQ ID NO: 292.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, having an IgG-scFv structure.
  • the second therapeutic agent as described in any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising two first chains of the structure represented by formula (a) and two formulas ( b) Second strand of the structure shown:
  • the second therapeutic agent of any one of the preceding items specifically binds PSMA and A bispecific antibody to CD3, which has an IgG-scFv structure, which includes two first chains of the structure represented by formula (a) and two second chains of the structure represented by formula (b):
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 313; and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 314; and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 315; and The CD3-VL includes the amino acid sequence of SEQ ID NO: 316.
  • the bispecific antibody that specifically binds PSMA and CD3 comprises two amino acid sequences of SEQ ID NO: 299 and two amino acid sequences of SEQ ID NO: 300.
  • the second therapeutic agent of any one of the preceding items is an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor targets PD-1 or CTLA4.
  • the second therapeutic agent of any one of the preceding items is an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
  • the second therapeutic agent of any one of the preceding items is an anti-PD-1 antibody, including any known therapeutically active anti-PD-1 antibody, including but not limited to camrelizumab (Camrelizumab), pembrolizumab (Keytruda), nivolumab (Opdivo) and cemiplimab (cemiplimab).
  • camrelizumab Camrelizumab
  • pembrolizumab Keytruda
  • nivolumab Opdivo
  • cemiplimab cemiplimab
  • the second therapeutic agent of any one of the preceding items is an anti-PD-1 antibody comprising a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL, wherein:
  • the PD-1-VH has: PD-1-HCDR1 including the amino acid sequence of SEQ ID NO: 319, PD-1-HCDR2 including the amino acid sequence of SEQ ID NO: 320 and the amino acid sequence including SEQ ID NO: 321 PD-1-HCDR3, and the PD-1-VL has: PD-1-LCDR1 comprising the amino acid sequence of SEQ ID NO: 322, PD-1-LCDR2 comprising the amino acid sequence of SEQ ID NO: 323 and PD-1-LCDR3 of the amino acid sequence of SEQ ID NO: 324.
  • the second therapeutic agent of any one is an anti-PD-1 antibody comprising a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL, wherein:
  • the PD-1-VH includes the amino acid sequence of SEQ ID NO: 325
  • the PD-1-VL includes the amino acid sequence of SEQ ID NO: 326.
  • the second therapeutic agent of any one of the preceding items is an anti-PD-1 antibody comprising a heavy chain of the amino acid sequence of SEQ ID NO: 328, and a light chain of the amino acid sequence of SEQ ID NO: 329 .
  • the antigen-binding molecules provided by the present disclosure have the characteristics of good therapeutic activity, safety, pharmacokinetic properties and druggability (such as stability).
  • Figure 1A Schematic structural diagram of Formula 1 of PSMA/CD28 bispecific antibody
  • FIG. 1B Schematic structural diagram of Formula 2 of PSMA/CD28 bispecific antibody
  • Figure 1C Schematic structural diagram of Formula 1 of PSMA/CD3 bispecific antibody
  • FIG. 1D Schematic structural diagram of Formula 5 of PSMA/CD3 bispecific antibody; where Ob represents Obscurin.
  • Figure 2 Fixed concentration of PSMA/CD3 bispecific antibody CC-1, killing results of different concentrations of PSMA/CD28 bispecific antibody on CHO-huPSMA cells.
  • Figure 3 Changes in the secretion of cytokine IFN- ⁇ in PBMCs under the combined action of PSMA/CD3 double antibodies, PSMA/CD28 double antibodies and CHO-huPSMA cells.
  • Figure 4 Changes in the secretion of cytokine IL-6 in PBMCs under the combined action of PSMA/CD3 double antibodies, PSMA/CD28 double antibodies and CHO-huPSMA cells.
  • CD28 cluster of differentiation 28, Tp44
  • CD28 refers to any CD28 protein from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., macaques) and rodents (e.g., mice and rats).
  • CD28 is expressed on T cells and provides costimulatory signals required for T cell activation and survival. In addition to T cell receptors (TCR), stimulation of T cells through CD28 can also provide effective signals for the production of various interleukins.
  • CD28 is the receptor for the CD80 (B7.1) and CD86 (B7.2) proteins and is the only B7 receptor constitutively expressed on naive T cells.
  • the amino acid sequence of human CD28 is shown in UniProt (www.uniprot.org) accession number P10747.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine.
  • Amino acid analogs have the same basic chemical structure as naturally occurring amino acids (i.e. alpha carbon bonded to hydrogen, carboxyl, amino and R groups) compounds such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium.
  • Such analogs have modified R groups (eg, norleucine) or modified peptide backbones but retain the same basic chemical structure as the naturally occurring amino acid.
  • Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of amino acids, but act in a manner similar to naturally occurring amino acids.
  • amino acid mutation includes amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to achieve the final construct, as long as the final construct possesses the desired properties, such as reduction or binding to Fc receptors.
  • Amino acid sequence deletions and insertions include deletions and insertions at the amino terminus and/or carboxyl terminus of the polypeptide chain.
  • Specific amino acid mutations may be amino acid substitutions.
  • the amino acid mutation is a non-conservative amino acid substitution, ie, one amino acid is replaced by another amino acid with different structural and/or chemical properties.
  • Amino acid substitutions include substitutions by non-naturally occurring amino acids or by derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine) .
  • Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is expected that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, may also be available. Various names may be used herein to refer to the same amino acid mutation.
  • the amino acid residue at a specific position can be represented by position + amino acid residue.
  • 366W means that the amino acid residue at position 366 is W.
  • T366W means that the amino acid residue at position 366 has been mutated from the original T to W.
  • antigen-binding molecule is used in the broadest sense and covers a variety of molecules that specifically bind to antigens, including but not limited to antibodies, other polypeptides with antigen-binding activity, and antibody fusion proteins formed by the fusion of the two, as long as they exhibit the desired Antigen-binding activity.
  • the antigen-binding molecules herein comprise a variable region (VH) and a variable region (VL), which together constitute an antigen-binding domain.
  • VH variable region
  • VL variable region
  • the antigen-binding molecule herein is a bispecific antigen-binding molecule (eg, bispecific antibody).
  • antibody is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibodies fragments (or antigen-binding fragments, or antigen-binding portions) as long as they exhibit the desired antigen-binding activity.
  • natural IgG antibodies are heterotetrameric proteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also known as variable heavy domain, heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C terminus, each light chain has a variable region (VL), also called a variable light domain, or light chain variable domain, followed by a constant light domain (light chain constant region, CL ).
  • VH variable region
  • CH1, CH2, and CH3 constant domain
  • bispecific antibody refers to an antibody (including antibodies or antigen-binding fragments thereof, such as single-chain antibodies) that can specifically bind to two different antigens or at least two different epitopes of the same antigen.
  • Bispecific antibodies of various structures have been disclosed in the prior art. According to the integrity of the IgG molecule, they can be divided into IgG-like bispecific antibodies and antibody fragment type bispecific antibodies. According to the number of antigen-binding regions, they can be divided into bivalent antibodies. , trivalent, quadrivalent or more valent bispecific antibodies, which can be divided into symmetric structure bispecific antibodies according to whether the structure is symmetrical or not. and asymmetrically structured bispecific antibodies.
  • bispecific antibodies based on antibody fragments such as Fab fragments lacking Fc fragments, which form bispecific antibodies by combining 2 or more Fab fragments in one molecule, have lower immunogenicity, and Small molecular weight, high tumor tissue penetration, typical antibody structures of this type such as F(ab)2, scFv-Fab, (scFv)2-Fab; IgG-like bispecific antibodies (for example, with Fc fragment),
  • This type of antibody has a relatively large molecular weight.
  • the Fc fragment helps purify the antibody and improves its solubility and stability.
  • the Fc part may also bind to the receptor FcRn, increasing the serum half-life of the antibody.
  • Typical bispecific antibody structural model Such as KiH, CrossMAb, Triomab quadroma, Fc ⁇ Adp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, 1Fab-IgG TDB, FynomAb, two-in-one/DAF, scFv-Fab-IgG , DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4 -Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immunol
  • variable region refers to the domain of an antigen-binding molecule that binds the antigen.
  • the heavy chain variable region in the antigen-binding module that specifically binds to PSMA is labeled PSMA-VH
  • the light chain variable region is labeled PSMA-VL
  • the heavy-chain variable region in the antigen-binding module that specifically binds to CD28 is labeled PSMA-VH.
  • CD28-VH the light chain variable region
  • CD28-VL each contain four conserved framework regions (FR) and three complementarity determining regions (CDR).
  • CDR complementarity determining region
  • framework or “FR” refers to the variable domain residues other than CDR residues.
  • VH contains 3 CDR areas: HCDR1, HCDR2 and HCDR3;
  • VL contains 3 CDR areas: LCDR1, LCDR2 and LCDR3.
  • the three CDR regions in PSMA-VH are labeled PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 respectively;
  • the three CDR regions in PSMA-VL are labeled PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 respectively.
  • CD28-VH The three CDR regions in CD28-VH are labeled CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 respectively; the three CDR regions in CD28-VL are labeled respectively CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3.
  • Each VH and VL from N end to C end are: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • a single VH or VL may be sufficient to confer antigen binding specificity.
  • the amino acid sequence boundaries of CDRs can be determined by various well-known schemes, such as: “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health , Bethesda, MD), "Chothia” numbering rule, “ABM” numbering rule, "contact” numbering rule (see Martin, ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001) and ImMunoGenTics (IMGT) numbering Rules (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278), etc.; the correspondence between various numbering systems is well known to those skilled in the art of.
  • the numbering scheme for this disclosure is set forth in Table 1 below.
  • variable region and CDR sequences in the embodiments of the present disclosure are all subject to the "Kabat" numbering rule.
  • one numbering system such as Kabat
  • Kabat is used to define amino acid residues
  • the corresponding technical solutions in other numbering systems will be regarded as equivalent technical solutions.
  • antibody fragment refers to a molecule other than an intact antibody that contains portions of an intact antibody that retain the antigen-binding ability of the intact antibody.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, single chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • Fc region or “fragment crystallizable region” is used to define the C-terminal region of an antibody heavy chain, including native Fc regions and engineered Fc regions.
  • the Fc region contains two subunits that are the same or different.
  • the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus.
  • Suitable native sequence Fc regions for use in the antibodies described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. Unless otherwise stated, the numbering convention for the Fc region is the EU index.
  • Titin chain refers to a peptide segment in the Titin protein containing a Titin Ig-like 152 domain or a functional variant thereof with a length of 78-118 amino acids.
  • the Titin chain can combine with the Obscurin chain to form a dimerization complex. things.
  • the term "Obscurin chain” refers to a peptide segment of the Obscurin protein containing the Obscurin Ig-like 1 domain or a functional variant thereof with a length of 87-117 amino acids, or a segment of the Obscurin-like 1 protein with a length of 78-118 amino acids.
  • a peptide segment of an amino acid containing an Obscurin-like Ig-like 1 domain or a functional variant thereof, and the Obscurin chain can combine with the Titin chain to form a dimerization complex.
  • the Titin chain and Obscurin chain disclosed in the present disclosure can be used to replace CH1 and CL in Fab to form a substituted Fab (Fab-S). This substitution does not affect the binding of the antigen-binding molecule to the antigen.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a specific source or species and the remaining portion of the heavy and/or light chain is derived from a different source or species.
  • humanized antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. This can be accomplished, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (ie, the constant regions as well as the framework portions of the variable regions).
  • affinity refers to the overall strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen).
  • binding affinity refers to the internal binding affinity that reflects a 1:1 between the members of a binding pair (e.g., antibody to antigen) interaction.
  • the affinity of a molecule X for its ligand Y can generally be expressed by the equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.
  • KD refers to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd/ka) and is expressed as molar concentration (M).
  • M molar concentration
  • the term "monoclonal antibody” refers to a population of antibodies or members thereof that are substantially homogeneous, ie, the antibody molecules contained in the population are identical in amino acid sequence, except for natural mutations that may be present in minor amounts. In contrast, polyclonal antibody preparations typically contain multiple different antibodies with different amino acid sequences in their variable domains, often specific for different epitopes. "Monoclonal” refers to the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.
  • antigen refers to a molecule or portion of a molecule capable of being selectively recognized or bound by an antigen-binding molecule (eg, an antibody).
  • An antigen may have one or more epitopes capable of interacting with different antigen-binding molecules (eg, antibodies).
  • epitope refers to an area or region on an antigen that is capable of specifically binding to an antibody or antigen-binding fragment thereof.
  • Epitopes may be formed from contiguous amino acids (linear epitopes) or contain non-contiguous amino acids (conformational epitopes), for example due to the folding of the antigen (i.e. the tertiary folding of the antigen by its proteinaceous nature) such that the non-contiguous amino acids are spatially separated. near.
  • the difference between conformational epitopes and linear epitopes is that in the presence of denaturing solvents, the antibody's binding to the conformational epitope is lost.
  • An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.
  • Screening for antibodies that bind a specific epitope can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443 -463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigen (see Prot.Sci.9 (2000) 487-496), and cross-blocking (see “Antibodies", Harlow and Lane (Cold Spring Harbor Press,Cold Spring Harb.,NY)).
  • the terms “capable of specifically binding”, “specifically binding” or “binding” refer to the ability of an antibody to bind to an antigen or epitope with higher affinity than to other antigens or epitopes.
  • antibodies bind an antigen or epitope with an equilibrium dissociation constant (KD) of about 1 ⁇ 10 ⁇ 7 M or less (eg, about 1 ⁇ 10 ⁇ 8 M or less).
  • KD equilibrium dissociation constant
  • the KD of the antibody binding to the antigen is 10% or less (eg, 1%) of the KD of the antibody binding to a non-specific antigen (eg, BSA, casein).
  • KD can be measured using known methods, such as by FACS or surface plasmon resonance assays.
  • antibodies that specifically bind to an antigen or its epitope may be cross-reactive to other related antigens, e.g., to antibodies from other species (homologous) such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus). , cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or marmoset (Callithrix jacchus) (commonmarmoset, marmoset) corresponding antigens are cross-reactive.
  • does not bind means that the antibody is unable to bind to an antigen or its epitope in the specific binding manner described above. For example, when the antibody binds the antigen or its epitope with an equilibrium dissociation constant (KD) of about 1 ⁇ 10 -6 M or greater.
  • KD equilibrium dissociation constant
  • antigen-binding module refers to a polypeptide molecule that specifically binds to a target antigen or an epitope thereof.
  • Specific antigen-binding modules include the antigen-binding domain of an antibody, for example, including a heavy chain variable region and a light chain variable region.
  • antigen-binding module that specifically binds CD28 refers to a module that is capable of binding CD28 or an epitope thereof with sufficient affinity such that molecules containing the module can be used as diagnostic and/or therapeutic agents targeting CD28.
  • an antigen-binding module that specifically binds CD28 has the following equilibrium dissociation constant (KD): ⁇ approximately 2 ⁇ 10 ⁇ 8 M, as measured by surface plasmon resonance assay.
  • Antigen binding moieties include antibody fragments as defined herein, such as Fab, substituted Fab or scFv.
  • linker refers to a linking unit that joins two polypeptide fragments.
  • linkers appearing in the same structural formula may be the same or different.
  • the linker can be a peptide linker, which contains one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids.
  • the linkers used herein may be the same or different.
  • Tm is the solution denaturation temperature (intrinsic fluorescence). When proteins are denatured (heated or denatured), the tertiary structure is opened and the aromatic amino acid microenvironment changes, resulting in a change in the emission fluorescence spectrum.
  • Tm1 refers to the temperature at which the fluorescence changes to half of its maximum value.
  • Tonset is the denaturation starting temperature. It means the temperature at which the protein begins to denature, that is, the temperature at which the fluorescence value begins to change.
  • Tagg is the aggregation onset temperature. By static light scattering, aggregates are detected at two wavelengths, 266 nm and 473 nm, and the temperature at which the sample begins to aggregate is monitored. Tagg 266 refers to the aggregation onset temperature monitored at 266nm.
  • nucleic acid is used interchangeably herein with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, naturally occurring and non-naturally occurring, have similar binding properties to the reference nucleic acid, and are Metabolized in a manner similar to the reference nucleotide.
  • nucleic acid molecules that has been separated from components of its natural environment.
  • Isolated nucleic acid encoding said antigen-binding molecule refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such one or more nucleic acids in a single vector or separate vectors molecule, and such one or more nucleic acid molecules present at one or more locations in a host cell.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences thereof as well as sequences explicitly indicated.
  • degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by a degenerate base and/or Deoxyinosine residue substitution.
  • polypeptide and "protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the term applies to amino acid polymers in which one or more amino acid residues are the corresponding artificial chemical mimetics of naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless stated otherwise, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • sequence identity refers to the extent (percentage) that the amino acids/nucleic acids of two sequences are identical at equivalent positions when the two sequences are optimally aligned. During the alignment process, gaps may be allowed to be introduced when necessary to achieve maximum percent sequence identity, but any conservative substitutions are not considered to form part of the sequence identity. To determine percent sequence identity, alignment can be accomplished by techniques known to those skilled in the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms required to achieve maximal alignment over the full length of the sequences being compared.
  • fusion means that the components (eg, the antigen-binding module and the Fc domain) are covalently linked, either directly or via a linker.
  • vector means a polynucleotide molecule capable of transporting another polynucleotide to which it is linked.
  • plasmid refers to a circular double-stranded DNA circle into which additional DNA segments can be ligated.
  • viral vector such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be ligated into the viral genome.
  • AAV adeno-associated viral vector
  • Certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors).
  • vectors eg, non-episomal mammalian vectors
  • expression vector or "expression construct” refers to a vector suitable for transformation of a host cell and containing the direction and/or control of the expression of one or more heterologous coding regions operably linked thereto (together with the host cell).
  • Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, RNA splicing of the coding region operably linked thereto.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages.
  • the progeny may not be identical in nucleic acid content to the parent cells, but may contain mutations.
  • mutant progeny that possess the same functional or biological activity as cells screened or selected in primary transformed cells.
  • Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell line plant cells, and fungal cells.
  • Mammalian host cells include human, mouse, rat, canine, monkey, porcine, goat, bovine, equine, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.
  • Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Cokra Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans), Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia spp., Saccharomycescerevisiae, Saccharomyces genus, Hansenula polymorpha, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans ), Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium
  • Pichia pastoris any Saccharomyces spp., Hansenula polymorpha, any Kluyveromyces spp., Candida albicans, any Aspergillus spp., Trichoderma reesei, Luck Chrysosporium lucknowense, any species of Fusarium, Yarrowia lipolytica and Neurospora crassa.
  • the host cells of this patent do not include subjects that are not authorized under the patent law.
  • composition means a mixture containing one or more antigen-binding molecules or antibodies described herein together with other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients.
  • pharmaceutically acceptable carrier refers to an ingredient of a pharmaceutical formulation that is distinct from the active ingredient and is not toxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • subject or “individual” includes humans and non-human animals.
  • Non-human animals include all vertebrates (eg, mammals and non-mammals) such as non-human primates (eg, cynomolgus monkeys), sheep, dogs, cattle, chickens, amphibians, and reptiles.
  • patient or “subject” are used interchangeably herein.
  • cyno or “cynomolgus” refers to the crab-eating monkey (Macaca fascicularis).
  • the individual or subject is a human.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, means the administration of an exogenous drug, therapeutic, diagnostic or composition to an animal, human , contact with subjects, cells, tissues, organs or biological fluids.
  • sample refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject.
  • exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph fluid, urine, saliva, cyst fluid, tears, excreta, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites , fluids from the pleura, pericardium, peritoneum, peritoneal cavity and other body cavities, fluids collected from bronchial lavage, synovial fluids, fluid solutions in contact with subjects or biological sources, For example, cell and organ culture media (including cell or organ conditioned media), lavage fluid, etc., tissue biopsy samples, fine needle aspiration, surgically resected tissue, organ culture or cell culture.
  • biological fluids such as blood, serum and serosal fluids, plasma, lymph fluid, urine, saliva, cyst fluid, tears, excreta, sputum, mucos
  • Treatment refers to a clinical intervention attempted to be applied to the individual being treated, and may be administered for preventive purposes, or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and regressing or improving prognosis .
  • molecules of the present disclosure are used to delay the development of a disease or slow the progression of a disease.
  • recurrence refers to the return of cancer or disease following clinical assessment of disease resolution.
  • diagnosis of distant cancer metastasis or local recurrence may be considered recurrence.
  • refractory or “resistant” refers to a cancer or disease that does not respond to treatment.
  • an “effective amount” is generally one sufficient to reduce the severity and/or frequency of symptoms, eliminate those symptoms and/or underlying causes, prevent the occurrence of symptoms and/or their underlying causes, and/or ameliorate or ameliorate impairments caused by or associated with the disease state. amount.
  • the effective amount is a therapeutically effective amount or a prophylactically effective amount.
  • a "therapeutically effective amount” is one sufficient to treat a disease state or symptom, particularly a condition or symptom associated with that disease state, or to otherwise prevent, hinder, delay or reverse the disease state or any other adverse effect in any way related to the disease. The ideal amount of symptomatic progression.
  • a “prophylactically effective amount” is an amount that, when administered to a subject, will have a predetermined prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. . Complete therapeutic or prophylactic effect does not necessarily occur after administration of one dose but may occur after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.
  • “Therapeutically effective amount” and “prophylactically effective amount” may vary depending on a variety of factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual.
  • Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.
  • Immune checkpoint means a group of molecules on the cell surface of CD4 T cells and CD8 T cells. These molecules can effectively act as “brakes” to downregulate or inhibit anti-tumor immune responses. Immune checkpoint molecules include, but are not limited to, programmed death 1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), B7H1, B7H4, OX-40, CD137, CD40, and LAG-3, which directly inhibit Immune Cells.
  • PD-1 programmed death 1
  • CTL-4 cytotoxic T lymphocyte antigen 4
  • B7H1, B7H4, OX-40 CD137, CD40, and LAG-3
  • Immune checkpoint inhibitors for use in the present disclosure are substances that inhibit the function of immune checkpoint molecules.
  • the immune checkpoint inhibitor is not particularly limited as long as the inhibitor is a substance that can inhibit the function (signal) of the immune checkpoint molecule.
  • Immune checkpoint inhibitors that can be used in the methods of the present disclosure include, but are not limited to, PD-1, PD-L2, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, Inhibitors of CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5) and/or TGFR ⁇ . Inhibition of inhibitory molecules can be effected by inhibition at the DNA, RNA or protein level.
  • inhibitory nucleic acids eg, dsRNA, siRNA, or shRNA
  • the inhibitor of an inhibitory signal is a polypeptide that binds to an inhibitory molecule, e.g., a soluble ligand or an antibody.
  • immune checkpoint inhibitors used in this disclosure can This includes, but is not particularly limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies, and anti-PD-1 antibodies and anti-PD-L1 antibodies may be preferred.
  • Antigen binding molecules of the present disclosure are provided.
  • the present disclosure provides antigen-binding molecules that have many advantageous properties, such as good in vitro killing activity, therapeutic activity, safety, pharmacokinetic properties and druggability (such as yield, purity and stability, etc.).
  • Antigen-binding molecules of the present disclosure include bispecific antigen-binding molecules (eg, bispecific antibodies) and anti-CD28 antibodies that specifically bind to CD28 and PSMA.
  • the antigen-binding molecules of the present disclosure have any of the following properties:
  • the anti-CD28 antibody binds human CD28 with an EC50 of less than 1 nM, as measured by ELISA.
  • the antigen-binding molecule activates IL-2-expressing Jurkat cells with an EC50 of less than 0.1 ⁇ g/mL in the presence of a first activation signal.
  • combination with PSMA/CD3 bispecific antibody or anti-PD-1 antibody has better in vivo therapeutic activity.
  • PSMA/CD3 bispecific antibody Combined with PSMA/CD3 bispecific antibody, it can significantly increase the killing effect of PSMA/CD3 bispecific antibody on tumor cells.
  • the present disclosure provides an antigen-binding molecule comprising at least one antigen-binding module that specifically binds to CD28 and at least one antigen-binding module that specifically binds to PSMA, the antigen-binding module that specifically binds to CD28 comprises CD28-VH and CD28 -VL, the antigen-binding module that specifically binds PSMA includes PSMA-VH and PSMA-VL.
  • the present disclosure also provides an anti-CD28 antibody capable of specifically binding to CD28, the antibody comprising CD28-VH and CD28-VL.
  • the Examples herein disclose antibody series 81, 94, 97, and 129. The antibodies of this article are described below using antibodies 97 and 94 as examples.
  • An antigen-binding molecule or anti-CD28 antibody that specifically binds CD28 and PSMA wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 22, as shown in SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 CD28-LCDR2 as shown and CD28-LCDR3 as shown in SEQ ID NO: 24; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 16, such as SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and CD28-LCDR3 as shown in SEQ ID NO: 18.
  • An antigen-binding molecule or anti-CD28 antibody that specifically binds CD28 and PSMA wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and
  • the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 22, CD28-LCDR2 as set forth in SEQ ID NO: 23, and CD28-LCDR3 as set forth in SEQ ID NO: 24; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and
  • the CD28-VL is CD28-LCDR1 as shown in SEQ ID NO: 16, CD28-LCDR2 as shown in SEQ ID NO: 62, and CD28-LCDR3 as shown in SEQ ID NO: 18.
  • the antigen-binding molecule or anti-CD28 antibody as described above, the CD28-VH and/or the CD28-VL is murine or humanized. In some embodiments, the CD28-VH and/or the CD28-VL are humanized.
  • the FR1, FR2, FR3, and FR4 of the humanized CD28-VH are at least 60%, 70%, 80%, or 90% identical to the FR1, FR2, FR3, and FR4 of SEQ ID NO:98 Sequence identity; FR1, FR2, FR3 and FR4 of the humanized CD28-VL have at least 60%, 70%, 80% or 90% identity with the FR1, FR2, FR3 and FR4 of SEQ ID NO: 102 Sequence identity.
  • the humanized CD28-VH has FR1, FR2, FR3 derived from IGHV1-46*01 and FR4 derived from IGHJ6*01, and is unsubstituted or has FR4 derived from IGHJ6*01 , one or more amino acid substitutions in the group consisting of 26A, 29L, 69L, 71V, 78A and 93S; and/or the humanized CD28-VL has FR1, FR2, FR3 derived from IGKV1-33*01 and FR4 derived from IGKJ4*01 and which is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44I and 71Y.
  • the amino acid positions in the variable regions described above are defined according to Kabat numbering rules.
  • the humanized CD28-VH comprises the amino acid sequence of SEQ ID NO: 98 or a variant thereof.
  • the variant is one or more amino acid substitutions in the FR region of SEQ ID NO: 98 selected from the group consisting of 26A, 29L, 69L, 71V, 78A, and 93S.
  • the humanized CD28-VL comprises the amino acid sequence of SEQ ID NO: 102, or a variant thereof.
  • the variant is one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44I, and 71Y in the FR region of SEQ ID NO: 102.
  • the FR1, FR2, FR3, and FR4 of the humanized CD28-VH are at least 60%, 70%, 80%, or 90% identical to the FR1, FR2, FR3, and FR4 of SEQ ID NO: 69 Sequence identity; FR1, FR2, FR3 and FR4 of the humanized CD28-VL have at least 60%, 70%, 80% or 90% identity with the FR1, FR2, FR3 and FR4 of SEQ ID NO:80 Sequence identity.
  • the humanized CD28-VH has FR1 derived from IGHV1-3*01, FR2, FR3 and FR4 derived from IGHJ1*01 and which are unsubstituted or have one or more amino acid substitutions selected from the group consisting of 1E, 28S, 69L, 71V, 73K and 94S; and/or the
  • the humanized CD28-VL has FR1, FR2, FR3 derived from IGKV1-12*01 and FR4 derived from IGKJ4*01, and is unsubstituted or has a group selected from the group consisting of 43S and 70K One or more amino acid substitutions.
  • the amino acid positions in the variable regions described above are defined according to Kabat numbering rules.
  • the humanized CD28-VH comprises the amino acid sequence of SEQ ID NO: 69 or a variant thereof.
  • the variant is one or more amino acid substitutions selected from the group consisting of 28S, 69L, 71V, 73K, and 94S in the FR region of SEQ ID NO: 69.
  • the humanized CD28-VL comprises the amino acid sequence of SEQ ID NO: 80, or a variant thereof.
  • the variant is one or more amino acid substitutions in the FR region of SEQ ID NO: 80.
  • the antigen-binding molecule or anti-CD28 antibody of any one of the above wherein the amino acid sequence of CD28-VH is at least 90% identical to SEQ ID NO: 95, 35, 94, 96, 97 or 98 , 95%, 96%, 97%, 98% or 99% sequence identity, and the amino acid sequence of CD28-VL is identical to SEQ ID NO: 101, 36, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115 have at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the CD28-VH has an amino acid sequence as set forth in SEQ ID NO: 95, 35, 94, 96, 97 or 98
  • the CD28-VL has an amino acid sequence as SEQ ID NO: 101, 36 , 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115.
  • the antigen-binding molecule or anti-CD28 antibody as described in any one of the preceding items the amino acid sequence of the CD28-VH is as shown in SEQ ID NO: 94, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115, or
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 95
  • the amino acid sequence of CD28-VL is shown in SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109 , 110, 111, 112, 113, 114 or 115, or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 96, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 99 or 100, or
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 97
  • the amino acid sequence of CD28-VL is shown in SEQ ID NO: 99, 100 or 101.
  • the antigen-binding molecule or anti-CD28 antibody as described in any one of the preceding items the amino acid sequence of the CD28-VH is as shown in SEQ ID NO: 95, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO:101 shown.
  • the antigen-binding molecule or anti-CD28 antibody of any one of the preceding items wherein the amino acid sequence of CD28-VH is consistent with SEQ ID NO: 68, 33, 63, 64, 65, 66, 67 or 69 Tool There is at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity, and the amino acid sequence of CD28-VL is identical to SEQ ID NO: 80, 34, 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79 have a sequence identity of at least 90%, 95%, 96%, 97%, 98% or 99%.
  • the CD28-VH has an amino acid sequence set forth in SEQ ID NO: 68, 33, 63, 64, 65, 66, 67, or 69
  • the CD28-VL has an amino acid sequence set forth in SEQ ID NO. :80, 34, 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79.
  • the antigen-binding molecule or anti-CD28 antibody as described in any one of the preceding items the amino acid sequence of the CD28-VH is as shown in SEQ ID NO: 63, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO:70 or 71 shown, or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 64, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 70, 71 or 72, or
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 65
  • the amino acid sequence of CD28-VL is shown in SEQ ID NO: 70, 71 or 72, or
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 66
  • the amino acid sequence of CD28-VL is shown in SEQ ID NO: 70, 71, 72, 74, 75, 76 or 79, or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 67
  • amino acid sequence of CD28-VL is shown in SEQ ID NO: 72, 74, 75, 76 or 79, or
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 68
  • the amino acid sequence of CD28-VL is shown in SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79 or 80 .
  • the antigen-binding molecule or anti-CD28 antibody as described in any one of the preceding items the amino acid sequence of the CD28-VH is as shown in SEQ ID NO: 68, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO:80 is shown.
  • the antigen-binding molecule as described above, wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and Said PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 167, PSMA-LCDR2 as shown in SEQ ID NO: 168, and PSMA-LCDR3 as shown in SEQ ID NO: 169; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 173, PSMA-LCDR2 as shown in SEQ ID NO: 174, and PSMA-LCDR3 as shown in SEQ ID NO: 175.
  • the antigen-binding molecule of any one of the preceding items, said PSMA-VH and/or said PSMA-VL is murine or humanized. In some embodiments, the PSMA-VH and/or the PSMA-VL are humanized. In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
  • amino acid sequence of PSMA-VH is shown in SEQ ID NO: 197, 199, 332, 194, 195, 196 or 198, and the amino acid sequence of PSMA-VL is SEQ ID NO: 201, 202, 333 or 200 shown; or
  • amino acid sequence of the PSMA-VH is shown in SEQ ID NO: 204, 184 or 203
  • amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 206, 185 or 205.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 202.
  • variable regions and CDRs described above are defined according to the Kabat numbering rule.
  • the antibody series 81 and 129 disclosed according to the embodiments herein have a similar technical solution scope to the antibody series 97 and 94 described above.
  • the bispecific antigen-binding molecules of the present disclosure are not limited to a specific molecular structure as long as they have the desired antigen-binding function.
  • the bispecific antigen-binding molecules herein may be bivalent (1+1), trivalent (2+1), or tetravalent (2+2).
  • the antigen-binding module in the antigen-binding molecule can be any antibody fragment with antigen-binding activity, which is fused through a peptide linker.
  • the peptide linkers of the present disclosure eg, linkers 1 to 11
  • the peptide linker can be a flexible peptide containing 1-50 or 3-20 amino acid residues.
  • the peptide linkers each independently have the structure of L 1 -(GGGGS)nL 2 , wherein L 1 is a bond, A, GS, GGS (SEQ ID NO: 399), GGGS (SEQ ID NO: 400), SGGGGS (SEQ ID NO: 294), GGGTKLTVLGGG (SEQ ID NO: 401), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is bond, G, GG, GGG (SEQ ID NO:295) or GGGG (SEQ ID NO:256), and the peptide linker is not a bond.
  • the peptide linker is 3-15 amino acid residues in length.
  • the peptide linkers each independently have the structure (GGGGS)n, where n is 1, 2, or 3.
  • the amino acid sequences of linker 1, linker 2, linker 3 and linker 4 are as shown in SEQ ID NO: 223.
  • the antigen-binding molecule of the present disclosure has a first chain with a structure represented by formula (a), a second chain with a structure represented by formula (b), and a second chain with a structure represented by formula (c-1).
  • antigen binding molecules include:
  • the antigen-binding molecule includes a first chain having a structure shown in formula (e-1), a second chain having a structure shown in formula (f-1), and a first chain having a structure shown in formula (g)
  • antigen binding molecules include:
  • amino acid sequence variants of the antigen-binding molecules provided herein are contemplated.
  • Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions, and/or substitutions of residues within the amino acid sequence of the antigen-binding molecule. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, so long as the final construct possesses the desired characteristics, such as antigen-binding properties.
  • antigen-binding molecule variants having one or more amino acid substitutions are provided. Substitutions can be made in CDR and FR. Conservative substitutions are shown in Table 2 under the heading "Preferred substitutions”. More substantial changes are provided in Table 2 under the heading "Exemplary Substitutions" and are described further below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the antibody of interest and the product screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
  • amino acids can be grouped as follows:
  • a non-conservative substitution would be the substitution of a member of one class for a member of another class.
  • substitution variant involves the substitution of one or more CDR residues of a parent antibody (eg, a humanized or human antibody).
  • a parent antibody eg, a humanized or human antibody
  • the resulting variants selected for further study will have alterations (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody, and/or will be substantially Retains certain biological properties of the parent antibody.
  • One exemplary substitution variant is an affinity matured antibody, which may be conveniently produced, for example, using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity).
  • Changes can be made to the CDRs, for example to improve antibody affinity.
  • affinity maturation diversity is introduced into the variable genes selected for maturation by any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis. middle. Then, create secondary libraries. The library is then screened to identify any antibody variants with the desired affinity.
  • CDR orientation in which several CDR residues (eg 4-6 residues at a time) are randomized.
  • CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling.
  • substitutions, insertions, or deletions may occur within one or more CDRs as long as such changes do not substantially reduce the ability of the antibody to bind the antigen.
  • conservative changes eg, conservative substitutions, as provided herein
  • each CDR is unchanged or contains no more than 1, 2, or 3 amino acid substitutions.
  • alanine scanning mutagenesis One method that can be used to identify residues or regions in an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis.”
  • a residue or group of residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g., Ala or polyalanine
  • Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution.
  • the contact points between the antibody and the antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include: fusion of 1 residue at the amino and/or carboxyl terminus or polypeptides of 100 or more residues in length; and intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include antibodies with an N-terminal methionyl residue.
  • Other insertional variants of antibody molecules include fusions with enzymes (or polypeptides that extend the serum half-life of the antibody) fused to the N- or C-terminus of the antibody.
  • one of the antigen-binding module that specifically binds PSMA and the antigen-binding module that specifically binds CD28 is a replaced Fab
  • the replaced Fab comprises Heavy chain variable region, light chain variable region, Titin chain and Obscurin chain.
  • the original CH1 and CL of the Fab are replaced by Titin chains and Obscurin chains.
  • the sequences of Titin chain and Obscurin chain are shown in Table 3-1 and Table 3-2.
  • the Fc region of an antigen-binding molecule of the present disclosure includes one or more amino acid substitutions that reduce its binding to an Fc receptor, e.g., its binding to an Fc ⁇ receptor, and reduce or Eliminate effector functions.
  • the native IgG Fc region specifically the IgG1 Fc region or the IgG4 Fc region, may cause the antigen-binding molecules of the present disclosure to target cells expressing Fc receptors rather than cells expressing the antigen.
  • the engineered Fc region of the present disclosure exhibits reduced binding affinity for Fc receptors and/or reduced effector function.
  • the engineered Fc region has a reduced binding affinity for the Fc receptor by more than 50%, 80%, 90%, or 95% compared to the native Fc region.
  • the Fc receptor is an Fc ⁇ receptor.
  • the Fc receptor is a human Fc ⁇ receptor, such as Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIB, Fc ⁇ RIIIa.
  • the engineered Fc region also has reduced binding affinity for complement, such as Clq, compared to the native Fc region.
  • the engineered Fc region has no reduced binding affinity for the neonatal Fc receptor (FcRn) compared to the native Fc region.
  • the engineered Fc region has reduced effector functions, which may include, but are not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced Antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced interaction with NK cells binding, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced of T cells.
  • CDC complement-dependent cytotoxicity
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • ADCP reduced antibody-dependent cellular phagocytosis
  • cytokine secretion reduced immune complex-mediated antigen uptake by antigen-presenting cells
  • reduced interaction with NK cells binding reduced binding to macrophages
  • monocytes reduced binding to monocytes
  • substitutions of amino acid residues at positions 238, 265, 269, 270, 297, 327, and 329 may reduce effector function.
  • the Fc region is a human IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index.
  • amino acid residue substitutions at positions such as 228 may reduce effector function.
  • Antigen-binding molecules may also contain disulfide bond modifications, such as 354C in the first subunit and 349C in the second subunit.
  • disulfide bond modifications such as 354C in the first subunit and 349C in the second subunit.
  • mutations 252Y, 254T, and 256E can be introduced.
  • the Fc region of the present disclosure includes modifications according to the knob-into-hole (KIH) technique, which involves introducing a knob at the interface of the first subunit and a knob at the interface of the second subunit.
  • KH knob-into-hole
  • a hole structure is introduced at the interface of the base. This allows the protruding structure to be positioned in the pore structure, promoting the formation of heterodimers and inhibiting the production of homodimers.
  • the bulge structure is built by replacing small amino acid side chains from the interface of the first subunit with larger side chains, such as tyrosine or tryptophan.
  • the pore structure is created in the interface of the second subunit by replacing large amino acid side chains with smaller amino acid side chains, such as alanine or threonine.
  • the bulge and pore structures are prepared by altering the nucleic acid encoding the polypeptide with optional amino acid substitutions as shown in the table below:
  • the C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; it can also be a truncated C-terminus, for example, one or two C-terminal amino acid residues have been removed from the truncated C-terminus.
  • the C-terminus of the heavy chain is a shortened C-terminus ending in PG.
  • a composition of intact antibodies may include a population of antibodies with all K447 residues and/or G446+K447 residues removed.
  • the composition of intact antibodies can include a population of antibodies without removal of the K447 residue and/or G446+K447 residues.
  • the composition of intact antibodies has a population of antibodies with and without a K447 residue and/or an antibody mixture of G446+K447 residues.
  • Antigen-binding molecules can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding the antigen-binding molecules are provided.
  • nucleic acids In the case of native antibodies, native antibody fragments or bispecific antibodies with homodimeric heavy chains, two nucleic acids are required, one for the light chain or fragments thereof and one for the heavy chain or fragments thereof.
  • nucleic acids encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH of the antibody (eg, the light chain and/or heavy chain of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.
  • nucleic acids are required, one for the first light chain, one for the first heavy chain comprising the first heterologous monomeric Fc region polypeptide, and one one for the second light chain, and one for the second heavy chain comprising a second heterologous monomeric Fc region polypeptide.
  • These four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors, usually these nucleic acids are located on two or three expression vectors, that is, one vector can contain more than one of these nucleic acids.
  • the present disclosure provides an isolated nucleic acid encoding an antibody as described above. Such nucleic acids can independently encode any of the aforementioned polypeptide chains.
  • the present disclosure provides one or more vectors (eg, expression vectors) comprising such nucleic acids.
  • the present disclosure provides host cells comprising such nucleic acids.
  • a method of preparing an antigen-binding molecule is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as provided above, and optionally The antibody is recovered from the host cell (or host cell culture medium).
  • nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and/or expression in host cells.
  • nucleic acids can be easily can be isolated and sequenced (for example, by using oligonucleotide probes capable of binding specifically to the genes encoding the antibody heavy and light chains), or produced by recombinant methods or obtained by chemical synthesis.
  • Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells described herein.
  • antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required for the antibody. After expression, the antibodies can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of vectors with Antibodies with partially or fully human glycosylation patterns.
  • Suitable host cells for expression of (glycosylated) antibodies may also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells.
  • baculovirus strains have been identified that can be used in combination with insect cells, especially for transfection of Spodoptera frugiperda cells; plant cell cultures can also be used as hosts, such as US5959177, US 6040498, US6420548, US7125978 and US6417429; vertebrate cells can also be used as hosts, such as mammalian cell lines adapted to growth in suspension.
  • Suitable mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7); the human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); mouse Sertoli ( sertoli) cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat (buffalo rat) liver cells ( BRL3A); human lung cells (W138); human liver cells (Hep G2); mouse breast tumors (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells.
  • COS-7 monkey kidney CV1 line
  • TM4 cells mouse Sertoli ( sertoli) cells
  • CV1 monkey kidney cells
  • VEO-76 African green monkey kidney cells
  • HELA human cervical cancer cells
  • BRL3A canine kidney cells
  • MDCK buffalo rat (buffalo rat) liver cells
  • W138 human liver cells
  • Hep G2
  • Suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines, such as Y0, NSO, and Sp2/0.
  • CHO Chinese hamster ovary
  • myeloma cell lines such as Y0, NSO, and Sp2/0.
  • the present disclosure provides antigen-binding molecules that can be used to detect the presence of PSMA and/or CD3 in a biological sample.
  • detection encompasses either quantitative or qualitative detection.
  • a biological sample includes cells or tissue, such as tumor tissue.
  • antigen binding molecules for use in diagnostic or detection methods are provided.
  • a method of detecting the presence of PSMA and/or CD3 in a biological sample is provided.
  • the method includes contacting a biological sample with an antigen-binding molecule under appropriate conditions and detecting whether a complex is formed between the detection reagent and the antigen.
  • antigen binding molecules are used to select subjects suitable for treatment, for example PSMA and/or CD3 are biomarkers used to select patients.
  • Exemplary conditions that can be diagnosed using the antigen-binding molecules of the present disclosure are, for example, tumors or cancers.
  • labeled antigen binding molecules include, but are not limited to, direct Labels or moieties that detect directly (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties that detect indirectly (e.g., moieties that detect indirectly via enzymatic reactions or molecular interactions, such as enzymes or ligands) body).
  • direct Labels or moieties that detect directly such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels
  • moieties that detect indirectly e.g., moieties that detect indirectly via enzymatic reactions or molecular interactions, such as enzymes or ligands
  • compositions comprising the antigen-binding molecules are provided, eg, for use in any of the following methods of treatment.
  • a pharmaceutical composition includes any of the antigen-binding molecules provided herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition includes any of the antigen-binding molecules provided herein and at least one additional therapeutic agent.
  • compositions of antigen-binding molecules of the present disclosure are prepared by mixing such antigen-binding molecules with the desired purity with one or more optional pharmaceutically acceptable carriers, the pharmaceutical compositions In the form of lyophilized compositions or aqueous solutions.
  • Formulations for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile membrane.
  • antigen-binding molecules Any of the antigen-binding molecules provided herein can be used in therapeutic methods.
  • the present disclosure provides use of an antigen-binding molecule in the manufacture or preparation of a medicament.
  • the medicament is used to treat proliferative diseases or tumors.
  • the drug is present in an effective amount for the above-mentioned diseases.
  • the effective amount is a unit daily dose or a unit weekly dose.
  • the use further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional treatments). agent).
  • a "subject" according to any of the above embodiments may be a human.
  • a pharmaceutical composition comprising the antigen-binding molecule, eg, for use in any of the above pharmaceutical uses or methods of treatment.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent.
  • the antigen-binding molecules of the present disclosure can be used alone or in combination with other agents for treatment.
  • the antigen-binding molecules of the present disclosure can be co-administered with at least one additional therapeutic agent.
  • the additional therapeutic agent is a bispecific antibody or an anti-PD-1 antibody that specifically binds PSMA and CD3.
  • the antigen-binding molecules of the present disclosure may be administered by any suitable means, including parenterally, intrapulmonary, and intranasal, and, if local treatment is desired, intralesional administration.
  • Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be by any appropriate route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term.
  • Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • the antigen-binding molecules of the present disclosure will be formulated, administered, and administered in a manner consistent with GOOD MEDICAL PRACTICE. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and others known to the medical practitioner factor.
  • the antigen-binding molecules need not be, but are optionally, formulated with one or more agents currently used to prevent or treat the disorder. Such other reagents
  • the effective amount depends on the amount of antigen-binding molecule present in the pharmaceutical composition, the type of condition or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as described herein, or at about 1 to 99% of the dosages described herein, or at any dosage and by any route empirically/clinically determined to be appropriate.
  • the antigen-binding molecules of the present disclosure when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the nature of the therapeutic molecule Type, severity and duration of disease, whether administration is for prophylactic or therapeutic purposes, previous treatments, patient's clinical history and response to therapeutic molecules, and the judgment of the attending physician.
  • the therapeutic molecules are appropriately administered to the patient in one session or over a series of treatments.
  • about 1 ⁇ g/kg to 15 mg/kg of the antigen-binding molecule may be an initial candidate dose for administration to the patient, whether, for example, by one or more divided administrations or by continuous infusion .
  • a typical daily dose may range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the exemplary unit daily dose is 50 ⁇ g-5g.
  • an article of manufacture contains materials useful in treating, preventing, and/or diagnosing the conditions described above.
  • the article includes a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like.
  • Containers can be formed from a variety of materials such as glass or plastic.
  • At least one active agent in the composition is an antigen-binding molecule of the present disclosure.
  • the label or package insert indicates use of the composition to treat the selected condition.
  • an article of manufacture may comprise: (a) a first container having a composition therein, wherein the composition comprises an antigen-binding molecule of the present disclosure; and (b) a second container having a composition therein, wherein the composition The agent contains additional cytotoxic agents or other therapeutic agents.
  • the article of manufacture in this embodiment of the present disclosure may further comprise a package insert indicating that the composition may be used to treat a particular condition.
  • the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer. It may further include other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
  • the article of manufacture is prepared in the form of a kit.
  • Titin chain/Obscurin chain of the present disclosure can be derived from any suitable polypeptide, including polypeptides derived from WO2021139758 (incorporated herein by reference in its entirety) and CN202110527339.7 and the patents (incorporated herein by reference in their entirety) which are priority documents. .
  • DI bispecific antibodies against hNGF and hRANKL are constructed: DI-2 to DI-20, which include the first heavy chain, the second heavy chain, the first light chain and the second Light chain:
  • the first heavy chain: from N-terminus to C-terminus is: [VH1-I]-[linker 1]-[Obscurin chain]-[Fc2],
  • the first light chain from N-terminal to C-terminal: [VL1-I]-[Linker 2]-[Titin chain],
  • Second heavy chain from N-terminus to C-terminus: [VH2-D]-[CH1]-[Fc1], and
  • Second light chain from N-terminus to C-terminus: [VL2-D]-[CL];
  • VH1-I and VL1-I are respectively the heavy chain variable region and light chain variable region of I0 in WO2021139758
  • VH2-D and VL2-D are respectively the heavy chain variable region and light chain variable region of D0 in WO2021139758. district.
  • the structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the DI bispecific antibody are shown in the table below.
  • Test Example 4 of WO2021139758 was used to detect the binding activity of the DI-2 to DI-20 bispecific antibodies and their antigens.
  • Thermal stability studies of antibodies were performed. Research method: Use PBS to dilute the concentration of the antibody to 5 mg/mL, and use a high-throughput differential scanning fluorometer (UNCHAINED, specification model: Unit) to measure its thermal stability. The experimental results showed that the antigen-binding activity of the modified bispecific antibody did not change significantly; and, compared with DI-2, the Tm1 of DI-4 to DI-8, DI-10 to DI-16, and DI-20 (°C) and Tonset (°C) are significantly improved, and the thermal stability of bispecific antibodies is better.
  • PL bispecific antibodies against hPDL1 and hCTLA4 were constructed: PL-1 to PL-19, which comprise the first heavy chain, the second heavy chain, the first light chain and the second light chain as follows:
  • the first heavy chain from N end to C end: [VH1-P]-[Linker 1]-[Obscurin chain]-[Fc1],
  • the first light chain from N-terminal to C-terminal: [VL1-P]-[Linker 2]-[Titin chain],
  • Second heavy chain from N-terminus to C-terminus: [VH2-L]-[CH1]-[Fc2], and
  • Second light chain from N-terminus to C-terminus: [VL2-L]-[CL];
  • VH1-P and VL1-P are the heavy chain variable region and light chain variable region of the h1831K antibody in WO2020177733A1, respectively.
  • the amino acid sequences of VH2-L and VL2-L are as follows.
  • the binding activity of the PL bispecific antibody was detected with reference to the ELISA method in Test Example 4 of WO2021139758, in which hPDL1 and hCTLA4 antigens were purchased from: Sino biology. Thermal stability studies of antibodies were performed. Method: Use PBS to dilute the concentration of the antibody to 1.4-3mg/mL, and use a high-throughput differential scanning fluorometer (UNCHAINED, specification model: Unit) to measure its thermal stability.
  • the experimental results show that the PL bispecific antibody still has good binding activity to the antigen; and, compared with PL-1, the Tm1 (°C), Tagg 266 (°C), Tonset (°C) of PL-2 to PL-19 There is a significant improvement, and the thermal stability of bispecific antibodies is better.
  • HJ bispecific antibodies against hIL5 and hTSLP were constructed: HJ-3 to HJ11, which comprise the first heavy chain, the second heavy chain, the first light chain and the second light chain as follows:
  • the first heavy chain: from N-terminal to C-terminal is: [VH1-H]-[Linker 1]-[Titin chain]-[Fc1],
  • the first light chain from N end to C end: [VL1-H]-[Linker 2]-[Obscurin chain],
  • Second heavy chain from N terminus to C terminus: [VH2-J]-[CH1]-[Fc2], and
  • Second light chain from N-terminus to C-terminus: [VL2-J]-[CL];
  • VH1-H and VL1-H are respectively the heavy chain variable region and light chain variable region of H0 in WO2021139758
  • VH2-J and VL2-J are respectively the heavy chain variable region and light chain variable region of J1 in WO2021139758. district.
  • the structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the HJ bispecific antibody in this example are shown in the table below.
  • the antigen-binding activity of the HJ bispecific antibody was detected with reference to the method in Test Example 4 in WO2021139758.
  • the method is: prepare the HJ bispecific antibody dilution solution with a buffer solution of 10mM acetic acid pH 5.5 and 9% sucrose, and then concentrate the bispecific antibody through ultrafiltration concentration to obtain different concentrations. HJ bispecific antibody solution (see Table 13-2 for the concentration of HJ bispecific antibody), and then place the concentrated solution in a 40°C incubator for incubation.
  • CHO-hPSMA Construct CHO-hPSMA, CHO-APC-hPSMA, CHO-hCD28, HEK293-hCD28, CHO-APC-hPSMA-PDL1 and Jurkat-PD1 cell lines.
  • the relevant protein sequences are as follows:
  • HEK293T cells co-transfect HEK293T cells (ATCC, CRL-11268) with three plasmids: pVSV-G, pCMV-dR8.91 and pCDH-hPSMA for packaging.
  • Virus 48 hours after transfection, virus-infected HEK293 (ATCC, CRL-1573), CHO-K1 (ATCC, CCL-61) or CHO-APC cells (Promega, JA9441) were collected. After two weeks of pressure screening, the cells were subdivided. After cloning, CHO-hPSMA, CHO-APC-hPSMA, CHO-hCD28 and HEK293-hCD28 cell lines with high expression of PSMA or CD28 were obtained through FACS detection.
  • the gene encoding human PDL1 or human PD1 full-length was cloned into the mammalian cell expression vector pCDH, and HEK293T cells were co-transfected with three plasmids: pVSV-G, pCMV-dR8.91 and pCDH-hPSMA ( CRL-11268) packaged virus, 48 hours after transfection, collect the virus to infect CHO-APC-hPSMA or Jurkat (ATCC, TIB-152) cells as mentioned above, and perform cell sorting through pressure screening to obtain high expression of PDL1 or PD1 CHO-APC-hPSMA-PDL1 and Jurkat-PD1 cell lines.
  • This disclosure uses hybridoma technology to prepare monoclonal antibodies against human CD28.
  • the resulting antibodies specifically bind to human CD28 with higher affinity, and can cross-bind with cynomolgus CD28 and bind to human CD28 and cynomolgus monkeys on the cell surface.
  • CD28 has good binding activity.
  • the initial immunization with protein is 50 ⁇ g, and the booster immunization is 25 ⁇ g each time.
  • Gold Adjuvant (Sigma Cat No.T2684) and Thermo Alum (Thermo Cat No. 77161) is an adjuvant for cross-immunization.
  • Cellular immunity was immunized according to 5E6 each time. After the initial immunization and seven boosting immunizations, mice with high serum antibody titers were selected for spleen cell fusion.
  • the immunogen sequence is as follows:
  • hybridoma culture supernatant was detected according to the hybridoma cell growth density. Hybridomas with good binding activity to human CD28 on the cell surface and good activation function were screened out. Monoclonal hybridoma cells were collected, RNA was extracted with NucleoZol (MN) (according to the kit instructions), and reverse transcription was performed (PrimeScript TM Reverse Transcriptase, Takara, cat#2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326Rev.B 0503) and then sequenced to obtain the amino acid sequence of the CDR and variable region of the antibody in the hybridoma cell line.
  • MN NucleoZol
  • variable region sequence of the mouse anti-CD28 antibody was combined with the heavy chain and light chain constant regions shown in SEQ ID NO: 144 and SEQ ID NO: 145 to obtain a chimeric antibody.
  • Chi81 represents a chimeric antibody comprising the m81 murine heavy chain variable region, the light chain variable region, and the heavy chain and light chain constant regions shown in SEQ ID NO: 144 and SEQ ID NO: 145. Chi94, Chi97 and Chi129 and so on.
  • FR1, FR2, and FR3 of IGKV1-12*01 and FR4 of IGKJ4*01 were selected as the light chain framework region template; FR1, FR2, FR3 of IGHV1-46*01, and IGHJ1*01 were selected.
  • FR4 serves as the heavy chain framework region template.
  • the amino acid residues at positions 43, 54 and/or 73 of the light chain variable region of the humanized antibody are substituted; and/or the amino acid residues at positions 1, 54 and/or 73 of the heavy chain variable region of the humanized antibody are substituted.
  • the amino acid residues at positions 69, 71, 73, 78, 94, 100a, 100b and/or 100c are substituted.
  • R71V means that according to the Kabat numbering system, R at position 71 is mutated to V; 100a means that according to the kabat numbering rules 100A bit, and so on.
  • the CDRs of the humanized antibody of m81 are as follows:
  • variable region of humanized antibody h81 is as follows: Note: The single underlined part is the CDR, the double underlined part is the amino acid substitution site, and the remaining parts are fr.
  • the humanized antibody of m94 selects FR1, FR2, FR3 of IGKV1-12*01, and FR4 of IGKJ4*01 as the light chain framework region template; selects FR1, FR2, FR3 of IGHV1-3*01 and FR4 of IGHJ1*01 As a heavy chain framework region template.
  • the amino acid residues at positions 43, 50, 51, 53, 54, 55 and/or 70 of the light chain variable region of the humanized antibody are substituted; and/or the heavy amino acid residues of the humanized antibody are substituted.
  • the amino acid residues at positions 1, 28, 66, 69, 71, 73 and/or 94 in the variable region of the chain are substituted.
  • R94S means that R at position 94 is mutated to S according to the Kabat numbering system.
  • the CDR region sequence of the humanized antibody of m94 is as follows:
  • the sequence of the humanized antibody h94 variable region is as follows: Note: The single underlined part is the CDR, the double underlined part is the amino acid substitution site, and the remaining parts are fr.
  • FR1, FR2, FR3 of IGKV1-33*01 and FR4 of IGKJ4*01 were selected as the light chain framework region template; FR1, FR2, FR3 and IGHJ6* of IGHV1-46*01 were selected.
  • FR4 of 01 serves as the heavy chain framework region template.
  • the amino acid residues at positions 41, 42, 43, 44, 50, 51, 52, 53, 54, 55, 56 and/or 71 on the light chain variable region of the humanized antibody are substituted; and/or substitution of amino acid residues at positions 1, 26, 29, 69, 71, 78 and/or 93 of the heavy chain variable region of the humanized antibody.
  • F71Y means that F at position 71 is mutated back to Y according to the Kabat numbering system.
  • the CDR regions of the m97 humanized antibody are as follows:
  • variable region of humanized antibody h97 is as follows: Note: The single underlined part is the CDR, the double underlined part is the amino acid substitution site, and the remaining parts are fr.
  • the humanized antibody of mouse antibody m129 selects FR1, FR2, FR3 of IGKV1-33*01, and FR4 of IGKJ4*01 as the light chain framework region template; selects FR1, FR2, FR3 and IGHJ6* of IGHV1-46*01 FR4 of 01 serves as the heavy chain framework region template.
  • the amino acid residues at positions 41, 42, 43, 44, 50, 53, 54, 55 and/or 73 of the light chain variable region of the humanized antibody are substituted; and/or human The amino acid residues at positions 1, 25, 30, 34, 52, 71 and/or 78 of the heavy chain variable region of the humanized antibody are substituted.
  • the CDRs of the humanized antibody to m129 are as follows:
  • variable region sequence of humanized antibody 129 is as follows:
  • the heavy chain variable region and light chain variable region of the anti-CD28 humanized antibody were recombined with the heavy chain constant region hIgG1:CH1-Fc and the light chain constant region CL, respectively, to obtain a full-length humanized antibody.
  • the humanized antibodies against CD28 of the present disclosure are as follows:
  • the full-length sequence of the anti-CD28 humanized antibody is as follows:
  • Control antibodies for anti-CD28 antibodies used in this disclosure are as follows:
  • REGN-hIgG1 was constructed with reference to patent US20190389951A1, and its sequence is as follows:
  • CD28 super agonist TGN1412 was prepared with reference to patent US07585960B2. The specific sequence is as follows:
  • the present disclosure prepares monoclonal antibodies against human PSMA through hybridoma technology.
  • the obtained antibody specifically binds to human PSMA with high affinity and can cross-react with cynomolgus monkey PSMA; the obtained antibody has good binding activity to human PSMA and cynomolgus monkey PSMA on the cell surface.
  • MN NucleoZol
  • the cDNA obtained by reverse transcription was PCR amplified and sequenced using mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503).
  • the amino acid sequences of the CDR and variable regions of the screened monoclonal hybridoma cell lines are as follows:

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Public Health (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne une molécule de Liaison à l'antigène se liant spécifiquement à PSMA et CD28 et son utilisation pharmaceutique. La molécule de liaison à l'antigène peut être utilisée pour traiter des tumeurs.
PCT/CN2023/087463 2022-04-11 2023-04-11 Molécule de liaison à l'antigène se liant spécifiquement à psma et cd28 et son utilisation pharmaceutique WO2023198015A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210371696.3 2022-04-11
CN202210371696 2022-04-11

Publications (1)

Publication Number Publication Date
WO2023198015A1 true WO2023198015A1 (fr) 2023-10-19

Family

ID=88328904

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/087463 WO2023198015A1 (fr) 2022-04-11 2023-04-11 Molécule de liaison à l'antigène se liant spécifiquement à psma et cd28 et son utilisation pharmaceutique

Country Status (2)

Country Link
TW (1) TW202405015A (fr)
WO (1) WO2023198015A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108699157A (zh) * 2016-01-14 2018-10-23 德国癌症研究公共权益基金会 Psma结合抗体及其用途
CN112351820A (zh) * 2018-06-21 2021-02-09 瑞泽恩制药公司 双特异性抗psma x抗cd28抗体及其用途
CN112513085A (zh) * 2018-05-24 2021-03-16 詹森生物科技公司 Psma结合剂及其用途
WO2021139758A1 (fr) * 2020-01-09 2021-07-15 江苏恒瑞医药股份有限公司 Nouveau complexe polypeptidique
CN113661177A (zh) * 2019-03-22 2021-11-16 里珍纳龙药品有限公司 EGFR x CD28多特异性抗体
WO2022007807A1 (fr) * 2020-07-07 2022-01-13 百奥泰生物制药股份有限公司 Anticorps bispécifique et son utilisation

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108699157A (zh) * 2016-01-14 2018-10-23 德国癌症研究公共权益基金会 Psma结合抗体及其用途
CN112513085A (zh) * 2018-05-24 2021-03-16 詹森生物科技公司 Psma结合剂及其用途
CN112351820A (zh) * 2018-06-21 2021-02-09 瑞泽恩制药公司 双特异性抗psma x抗cd28抗体及其用途
CN113661177A (zh) * 2019-03-22 2021-11-16 里珍纳龙药品有限公司 EGFR x CD28多特异性抗体
WO2021139758A1 (fr) * 2020-01-09 2021-07-15 江苏恒瑞医药股份有限公司 Nouveau complexe polypeptidique
WO2022007807A1 (fr) * 2020-07-07 2022-01-13 百奥泰生物制药股份有限公司 Anticorps bispécifique et son utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YOU GIHOON, WON JONGHWA, LEE YANGSOON, MOON DAIN, PARK YUNJI, LEE SANG HOON, LEE SEUNG-WOO: "Bispecific Antibodies: A Smart Arsenal for Cancer Immunotherapies", VACCINES, vol. 9, no. 7, pages 724, XP093000326, DOI: 10.3390/vaccines9070724 *

Also Published As

Publication number Publication date
TW202405015A (zh) 2024-02-01

Similar Documents

Publication Publication Date Title
CN111744013B (zh) 抗tigit抗体联合pd-1抑制剂治疗疾病的方法和药物组合
TWI830151B (zh) 抗GPRC5DxBCMAxCD3三特異性抗體及其用途
WO2023198015A1 (fr) Molécule de liaison à l'antigène se liant spécifiquement à psma et cd28 et son utilisation pharmaceutique
WO2023198042A1 (fr) Molécule de liaison à l'antigène se liant spécifiquement à egfr et cd28, et son utilisation médicale
WO2024027815A1 (fr) Molécule de liaison à l'antigène se liant spécifiquement à gucy2c et cd3 et son utilisation pharmaceutique
WO2023174238A1 (fr) Molécule de liaison à l'antigène se liant spécifiquement à gprc5d et cd3 et son utilisation médicale
WO2023246885A1 (fr) Molécule de liaison à l'antigène se liant spécifiquement à dll3 et cd3, et son utilisation pharmaceutique
IL299232A (en) Bispecific antibody and its use
WO2023147784A1 (fr) Molécule de liaison à l'antigène se liant spécifiquement à psma et à cd3, et son utilisation pharmaceutique
WO2022156739A1 (fr) Molécule de liaison à l'antigène se liant spécifiquement à bcma et cd3 et son utilisation médicale
WO2023284806A1 (fr) Molécule de liaison à l'antigène qui se lie spécifiquement à cd38, bcma et cd3 et ses utilisations médicales
TW202413416A (zh) 特異性結合ｅｇｆｒ和ｃｄ２８的抗原結合分子及其醫藥用途
WO2024051804A1 (fr) Anticorps anti-ilt4 et utilisation pharmaceutique associée
WO2023051786A1 (fr) Molécules de liaison à l'antigène qui se lient spécifiquement à cgrp et pacap et leur utilisation pharmaceutique
TW202413437A (zh) 特異性結合ｇｕｃｙ２ｃ和ｃｄ３的抗原結合分子及其醫藥用途
WO2023165514A1 (fr) Molécule de liaison à l'antigène se liant spécifiquement à flt3 et cd3 et son utilisation pharmaceutique
WO2023284829A1 (fr) Molécule de liaison à l'antigène se liant spécifiquement à hgfr et eger, et utilisation pharmaceutique de celle-ci
WO2022237882A1 (fr) Molécule de liaison à l'antigène
WO2023083298A1 (fr) Protéine de fusion d'anticorps anti-icosl et son utilisation
WO2023208182A1 (fr) Anticorps anti-ccr8 et son utilisation
CA3232472A1 (fr) Anticorps anti-klb et leurs utilisations
TW202413414A (zh) 抗ｉｌｔ４抗體及其醫藥用途
CN115386008A (zh) 特异性结合cd20和cd3的抗原结合分子及其医药用途
KR20240067092A (ko) 항-klb 항체 및 용도

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23787659

Country of ref document: EP

Kind code of ref document: A1